rabbit anti xrn1 antibody affinity purified  (Bethyl)

 
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    Structured Review

    Bethyl rabbit anti xrn1 antibody affinity purified
    SMG6-mediated endonucleolytic cleavage is impaired when CASC3 is not present. ( A ) Schematic depiction of the globin mRNA reporter. The reporter consists of three exons (orange boxes) followed by an <t>XRN1-resistant</t> element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the second exon. ( B ) Northern blot of RNA extracted from the indicated cell lines that stably express the globin reporter. The xrFrag corresponds to the 3′ part of the reporter that is resistant to degradation by XRN1 due to the xrRNA. The cell lines in lanes 3 and 6 were additionally treated with XRN1 siRNA which results in the appearance of a 3′ degradation fragment below the full-length reporter. Reporter and 3′ fragment mRNA levels were normalized to 7SL RNA which is shown as a loading control. For the relative mRNA quantification, in each condition (WT versus CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the globin WT reporter (lanes 1 and 4). Individual data points and means are plotted from n = 3 experiments. ( C ) Schematic depiction of a TOE1 minigene reporter consisting of exons 6–8 (purple boxes) followed by a probe binding cassette (gray boxes). The reporter contains either the canonical stop codon (TOE1-WT) or PTCs in exon 7 (TOE1-PTC). ( D ) Northern blot of RNA extrac ted from the indicated cell lines treated with the indicated siRNAs stably expressing the TOE1 reporter depicted in Figure 5C . The intron between exon 6 and 7 is deleted so that the reporter is constitutively spliced to contain either a normal stop codon (TOE1-WT) or a PTC (TOE1-PTC). The mRNA levels were normalized to 7SL RNA. For the relative mRNA quantification, in each condition (WT versus CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the TOE-WT reporter (lanes 1 and 4). ( E ) Schematic depiction of the triose phosphate isomerase (TPI) mRNA reporter. The reporter consists of seven exons (blue boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the fifth exon. ( F ) Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing the either the TPI WT or TPI PTC mRNA reporter. ( G ) Quantification of the northern blot shown in Figure 5F . The reporter and 3′ fragment mRNA levels were normalized to the 7SL control. For each cell line, the mRNA levels were then normalized to the respective TPI WT reporter or 3′ fragment levels. Individual data points and means are plotted from n = 3 experiments.
    Rabbit Anti Xrn1 Antibody Affinity Purified, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex"

    Article Title: CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa564

    SMG6-mediated endonucleolytic cleavage is impaired when CASC3 is not present. ( A ) Schematic depiction of the globin mRNA reporter. The reporter consists of three exons (orange boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the second exon. ( B ) Northern blot of RNA extracted from the indicated cell lines that stably express the globin reporter. The xrFrag corresponds to the 3′ part of the reporter that is resistant to degradation by XRN1 due to the xrRNA. The cell lines in lanes 3 and 6 were additionally treated with XRN1 siRNA which results in the appearance of a 3′ degradation fragment below the full-length reporter. Reporter and 3′ fragment mRNA levels were normalized to 7SL RNA which is shown as a loading control. For the relative mRNA quantification, in each condition (WT versus CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the globin WT reporter (lanes 1 and 4). Individual data points and means are plotted from n = 3 experiments. ( C ) Schematic depiction of a TOE1 minigene reporter consisting of exons 6–8 (purple boxes) followed by a probe binding cassette (gray boxes). The reporter contains either the canonical stop codon (TOE1-WT) or PTCs in exon 7 (TOE1-PTC). ( D ) Northern blot of RNA extrac ted from the indicated cell lines treated with the indicated siRNAs stably expressing the TOE1 reporter depicted in Figure 5C . The intron between exon 6 and 7 is deleted so that the reporter is constitutively spliced to contain either a normal stop codon (TOE1-WT) or a PTC (TOE1-PTC). The mRNA levels were normalized to 7SL RNA. For the relative mRNA quantification, in each condition (WT versus CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the TOE-WT reporter (lanes 1 and 4). ( E ) Schematic depiction of the triose phosphate isomerase (TPI) mRNA reporter. The reporter consists of seven exons (blue boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the fifth exon. ( F ) Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing the either the TPI WT or TPI PTC mRNA reporter. ( G ) Quantification of the northern blot shown in Figure 5F . The reporter and 3′ fragment mRNA levels were normalized to the 7SL control. For each cell line, the mRNA levels were then normalized to the respective TPI WT reporter or 3′ fragment levels. Individual data points and means are plotted from n = 3 experiments.
    Figure Legend Snippet: SMG6-mediated endonucleolytic cleavage is impaired when CASC3 is not present. ( A ) Schematic depiction of the globin mRNA reporter. The reporter consists of three exons (orange boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the second exon. ( B ) Northern blot of RNA extracted from the indicated cell lines that stably express the globin reporter. The xrFrag corresponds to the 3′ part of the reporter that is resistant to degradation by XRN1 due to the xrRNA. The cell lines in lanes 3 and 6 were additionally treated with XRN1 siRNA which results in the appearance of a 3′ degradation fragment below the full-length reporter. Reporter and 3′ fragment mRNA levels were normalized to 7SL RNA which is shown as a loading control. For the relative mRNA quantification, in each condition (WT versus CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the globin WT reporter (lanes 1 and 4). Individual data points and means are plotted from n = 3 experiments. ( C ) Schematic depiction of a TOE1 minigene reporter consisting of exons 6–8 (purple boxes) followed by a probe binding cassette (gray boxes). The reporter contains either the canonical stop codon (TOE1-WT) or PTCs in exon 7 (TOE1-PTC). ( D ) Northern blot of RNA extrac ted from the indicated cell lines treated with the indicated siRNAs stably expressing the TOE1 reporter depicted in Figure 5C . The intron between exon 6 and 7 is deleted so that the reporter is constitutively spliced to contain either a normal stop codon (TOE1-WT) or a PTC (TOE1-PTC). The mRNA levels were normalized to 7SL RNA. For the relative mRNA quantification, in each condition (WT versus CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the TOE-WT reporter (lanes 1 and 4). ( E ) Schematic depiction of the triose phosphate isomerase (TPI) mRNA reporter. The reporter consists of seven exons (blue boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the fifth exon. ( F ) Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing the either the TPI WT or TPI PTC mRNA reporter. ( G ) Quantification of the northern blot shown in Figure 5F . The reporter and 3′ fragment mRNA levels were normalized to the 7SL control. For each cell line, the mRNA levels were then normalized to the respective TPI WT reporter or 3′ fragment levels. Individual data points and means are plotted from n = 3 experiments.

    Techniques Used: Binding Assay, Northern Blot, Stable Transfection, Expressing

    The CASC3 N-terminus promotes but is not necessary to elicit NMD. ( A ) Schematic depiction of the TPI-4MS2V5-SMG5 tethering reporter. The reporter consists of the TPI ORF (blue boxes) followed by 4 MS2 stem loops (SL). Downstream the SMG5 3′ untranslated region (UTR) is inserted to increase the size of 3′ fragments that result from cleavage at the termination codon. Reporter and 3′ fragment mRNAs can be detected via the probe binding cassette (gray boxes). ( B ) Northern blot of a tethering assay performed in HeLa Tet-Off cells. The cells stably express the tethering reporter shown in Figure 6A together with the indicated MS2V5-tagged proteins. When the cells are additionally treated with XRN1 siRNA, a 3′ degradation fragment can be detected below the full-length reporter. The reporter and 3′ fragment mRNA levels are normalized to the 7SL RNA. For the calculation of the relative mRNA levels in each condition (Luc vs. XRN1) the levels were normalized to the MS2V5-GST control (lanes 1 and 5). ( C ) Schematic depiction of CASC3 rescue protein constructs. The full-length (FL) protein consists of an N-terminal (blue), C-terminal (orange) and central SELOR domain (purple). The construct 1–480 has a C-terminal deletion, whereas in the construct 110–480 both the N- and C-terminus are truncated. Both deletion constructs were also rendered EJC-binding deficient by mutating the amino acid residues 188 and 218 (F188D, W218D). ( D ) Relative quantification of the CLN6 (top) and TOE1 (bottom) transcript isoforms by qPCR in the indicated cell lines. The V5-tagged rescue proteins expressed in the KO condition are shown schematically in Figure 6C . Rescue protein expression is confirmed in E and F. Individual data points and means are plotted from n = 3 experiments. (E and F) Western blot of samples shown in Figure 6D . The expression of rescue proteins was confirmed by an antibody against CASC3 ( E ) and an antibody recognizing the V5 tag ( F ).
    Figure Legend Snippet: The CASC3 N-terminus promotes but is not necessary to elicit NMD. ( A ) Schematic depiction of the TPI-4MS2V5-SMG5 tethering reporter. The reporter consists of the TPI ORF (blue boxes) followed by 4 MS2 stem loops (SL). Downstream the SMG5 3′ untranslated region (UTR) is inserted to increase the size of 3′ fragments that result from cleavage at the termination codon. Reporter and 3′ fragment mRNAs can be detected via the probe binding cassette (gray boxes). ( B ) Northern blot of a tethering assay performed in HeLa Tet-Off cells. The cells stably express the tethering reporter shown in Figure 6A together with the indicated MS2V5-tagged proteins. When the cells are additionally treated with XRN1 siRNA, a 3′ degradation fragment can be detected below the full-length reporter. The reporter and 3′ fragment mRNA levels are normalized to the 7SL RNA. For the calculation of the relative mRNA levels in each condition (Luc vs. XRN1) the levels were normalized to the MS2V5-GST control (lanes 1 and 5). ( C ) Schematic depiction of CASC3 rescue protein constructs. The full-length (FL) protein consists of an N-terminal (blue), C-terminal (orange) and central SELOR domain (purple). The construct 1–480 has a C-terminal deletion, whereas in the construct 110–480 both the N- and C-terminus are truncated. Both deletion constructs were also rendered EJC-binding deficient by mutating the amino acid residues 188 and 218 (F188D, W218D). ( D ) Relative quantification of the CLN6 (top) and TOE1 (bottom) transcript isoforms by qPCR in the indicated cell lines. The V5-tagged rescue proteins expressed in the KO condition are shown schematically in Figure 6C . Rescue protein expression is confirmed in E and F. Individual data points and means are plotted from n = 3 experiments. (E and F) Western blot of samples shown in Figure 6D . The expression of rescue proteins was confirmed by an antibody against CASC3 ( E ) and an antibody recognizing the V5 tag ( F ).

    Techniques Used: Binding Assay, Northern Blot, Stable Transfection, Construct, Real-time Polymerase Chain Reaction, Expressing, Western Blot

    2) Product Images from "Genome-wide analyses of XRN1-sensitive targets in osteosarcoma cells identifies disease-relevant transcripts containing G-rich motifs"

    Article Title: Genome-wide analyses of XRN1-sensitive targets in osteosarcoma cells identifies disease-relevant transcripts containing G-rich motifs

    Journal: bioRxiv

    doi: 10.1101/2020.08.11.246249

    XRN1 also regulates ncRNAs in SAOS-2 cells. A) Assessment of transcript proportions affected by XRN1 depletion relative to the genome wide proportions detected in our sequencing data. HOTAIR highlighted in black, grey=no change, red=post-transcriptionally upregulated, orange=transcriptionally upregulated, purple=post-transcriptionally downregulated, blue=transcriptionally downregulated, green=no intron data. B) Scatter plot of changes in expression of all ncRNAs detected in our sequencing data. C-E) Boxplots of C) expression D) GC content and E) length (bp) of ncRNAs in our data. Grouped by their nature of change in expression and compared to the genome average as detected in our data set.
    Figure Legend Snippet: XRN1 also regulates ncRNAs in SAOS-2 cells. A) Assessment of transcript proportions affected by XRN1 depletion relative to the genome wide proportions detected in our sequencing data. HOTAIR highlighted in black, grey=no change, red=post-transcriptionally upregulated, orange=transcriptionally upregulated, purple=post-transcriptionally downregulated, blue=transcriptionally downregulated, green=no intron data. B) Scatter plot of changes in expression of all ncRNAs detected in our sequencing data. C-E) Boxplots of C) expression D) GC content and E) length (bp) of ncRNAs in our data. Grouped by their nature of change in expression and compared to the genome average as detected in our data set.

    Techniques Used: Genome Wide, Sequencing, Expressing

    Direct XRN1 targets possess G-rich motifs. A/B) MEME analysis of 3’UTR of 103 mRNAs reveal A) GU rich 18 sites across 10 unique transcripts and B) G-rich 233 sites across 69 unique transcripts, motifs which may confer XRN1 sensitivity. C) Similar analysis of 30 ncRNAs post-transcriptionally upregulated in XRN1-depleted SAOS-2 cells reveals a similar G-rich motif to that observed in B (89 sites across 21 unique transcripts).
    Figure Legend Snippet: Direct XRN1 targets possess G-rich motifs. A/B) MEME analysis of 3’UTR of 103 mRNAs reveal A) GU rich 18 sites across 10 unique transcripts and B) G-rich 233 sites across 69 unique transcripts, motifs which may confer XRN1 sensitivity. C) Similar analysis of 30 ncRNAs post-transcriptionally upregulated in XRN1-depleted SAOS-2 cells reveals a similar G-rich motif to that observed in B (89 sites across 21 unique transcripts).

    Techniques Used:

    XRN1 is downregulated in osteo- and Ewing sarcoma. A) qRT-PCR quantification of XRN1 mRNA expression across osteosarcoma (OS) cell lines in comparison to the HOb control cell line, normalised to HPRT1 . Error bars represent SEM, n≥5. B) qRT-PCR quantification of pre-XRN1 across osteosarcoma cell lines in comparison to the HOb control cell line, normalised to HPRT1 . Error bars represent SEM, n≥6, C) qRT-PCR quantification of XRN1 across osteosarcoma patient samples in comparison to the HOb control cell line, normalised to PES1 . Error bars represent SEM, n≥5, p=0.0296. Red = samples from hip and femur, blue=samples from scapula or humerus and green=unknown origin. D) Representative Western blot and graphical analysis showing expression of XRN1 protein in osteosarcoma cells as a proportion of that expressed in HOb control cells. Error bars represent SEM, n≥4. E) qRT-PCR quantification of XRN1 mRNA expression in Ewing sarcoma (EWS) cell lines in comparison to the HOb control cell line, normalised to GAPDH . Error bars represent SEM, n≥6. F) Representative Western blot and graphical analysis showing expression of XRN1 protein in Ewing sarcoma cells as a proportion of that expressed in Hob control cells Error bars represent SEM, n≥4. For all figures ****=p
    Figure Legend Snippet: XRN1 is downregulated in osteo- and Ewing sarcoma. A) qRT-PCR quantification of XRN1 mRNA expression across osteosarcoma (OS) cell lines in comparison to the HOb control cell line, normalised to HPRT1 . Error bars represent SEM, n≥5. B) qRT-PCR quantification of pre-XRN1 across osteosarcoma cell lines in comparison to the HOb control cell line, normalised to HPRT1 . Error bars represent SEM, n≥6, C) qRT-PCR quantification of XRN1 across osteosarcoma patient samples in comparison to the HOb control cell line, normalised to PES1 . Error bars represent SEM, n≥5, p=0.0296. Red = samples from hip and femur, blue=samples from scapula or humerus and green=unknown origin. D) Representative Western blot and graphical analysis showing expression of XRN1 protein in osteosarcoma cells as a proportion of that expressed in HOb control cells. Error bars represent SEM, n≥4. E) qRT-PCR quantification of XRN1 mRNA expression in Ewing sarcoma (EWS) cell lines in comparison to the HOb control cell line, normalised to GAPDH . Error bars represent SEM, n≥6. F) Representative Western blot and graphical analysis showing expression of XRN1 protein in Ewing sarcoma cells as a proportion of that expressed in Hob control cells Error bars represent SEM, n≥4. For all figures ****=p

    Techniques Used: Quantitative RT-PCR, Expressing, Western Blot

    Gene Ontology analysis of differentially expressed transcripts. A) Gene ontology analysis using DAVID and Biological processes level “BPFAT” at highest stringency on all differentially expressed transcripts in XRN1-depleted SAOS-2 cells. B) As A, but enriched biological processes assessed in individual groups of misregulated transcripts.
    Figure Legend Snippet: Gene Ontology analysis of differentially expressed transcripts. A) Gene ontology analysis using DAVID and Biological processes level “BPFAT” at highest stringency on all differentially expressed transcripts in XRN1-depleted SAOS-2 cells. B) As A, but enriched biological processes assessed in individual groups of misregulated transcripts.

    Techniques Used:

    XRN1-sensitive transcripts show specific transcript characteristics. Boxplots of A) expression, B) GC content, C) translational efficiency, D) 5’UTR length, E) coding sequence (CDS) length and F) 3’UTR length of all transcripts within our data set. Grouped by their nature of change in expression and compared to the genome average as detected in our data set. Translational efficiency calculated as ribosome protected footprint FPKM/total RNA FPKM for each transcript.
    Figure Legend Snippet: XRN1-sensitive transcripts show specific transcript characteristics. Boxplots of A) expression, B) GC content, C) translational efficiency, D) 5’UTR length, E) coding sequence (CDS) length and F) 3’UTR length of all transcripts within our data set. Grouped by their nature of change in expression and compared to the genome average as detected in our data set. Translational efficiency calculated as ribosome protected footprint FPKM/total RNA FPKM for each transcript.

    Techniques Used: Expressing, Sequencing

    Overview of RNA-sequencing of XRN1-depleted SAOS-2 cells. A) Up- (red) and downregulated (blue) transcripts based on initial edgeR differential expression using genes as a counting method in featureCounts. B) Scrambled vs XRN1 knockdown FPKM based demonstrating differentially expressed transcripts using both gene and exon counting. Exon FPKM used for direct comparison with intron counting. Grey=no change, red=post-transcriptionally upregulated, orange=transcriptionally upregulated, purple=post-transcriptionally downregulated, blue=transcriptionally downregulated, green=no intron data. C) Differentially expressed transcripts when counting intron mapping reads allowing differentiation between transcriptional and post-transcriptional changes represented in B. Legend as in B. D) MA plot representing fold change in XRN1-depeleted SAOS-2 cells vs transcript expression in control cells coloured by nature of change. Legend as in B. E) Volcano plot demonstrating statistical information of all expressed transcripts. Legend as in B.
    Figure Legend Snippet: Overview of RNA-sequencing of XRN1-depleted SAOS-2 cells. A) Up- (red) and downregulated (blue) transcripts based on initial edgeR differential expression using genes as a counting method in featureCounts. B) Scrambled vs XRN1 knockdown FPKM based demonstrating differentially expressed transcripts using both gene and exon counting. Exon FPKM used for direct comparison with intron counting. Grey=no change, red=post-transcriptionally upregulated, orange=transcriptionally upregulated, purple=post-transcriptionally downregulated, blue=transcriptionally downregulated, green=no intron data. C) Differentially expressed transcripts when counting intron mapping reads allowing differentiation between transcriptional and post-transcriptional changes represented in B. Legend as in B. D) MA plot representing fold change in XRN1-depeleted SAOS-2 cells vs transcript expression in control cells coloured by nature of change. Legend as in B. E) Volcano plot demonstrating statistical information of all expressed transcripts. Legend as in B.

    Techniques Used: RNA Sequencing Assay, Expressing

    XRN1 knockdown in SAOS-2 cells does not result in observable phenotypes. A) Successful knockdown of XRN1 in SAOS-2 cells using RNAi 24 hours post transfection. Scr samples treated with 20pmol scrambled siRNA and KD cells treated with 20pmol XRN1 siRNA. Error bars represent SEM, ***=p=0.0008. B) Quantification and representative images (40x objective) of the BrdU proliferation assay. Error bars represent SEM, n≥25, p=0.7938, scale bar=50µM. C) WST-1 assay at 24hr time intervals following transfection with either Scrambled (Scr) or XRN1 (KD) siRNA. Error bar represent SEM, n=3. D) Caspase Glo 3/4 assay at 24hr time intervals following transfection with either Scrambled (Scr) or XRN1 (KD) siRNA. Error bar represent SEM, n=3. E) Quantification and representative images (20x objective) of transwell migration assay 6hrs, 24hrs or 30hrs post seeding. Seeding was performed 24hrs post transfection with either Scrambled (Scr) or XRN1 (KD) siRNA. Error bars represent SEM, n=4, p > 0.05, scale bar=100µM. F) Knockdown of XRN1 does not affect nascent translation rates. Quantification of Puromycin incorporation or XRN1 expression (normalised to GAPDH relative to its own scrambled partner) 24hrs post transfection in cells treated with either Scrambled (Scr) or XRN1 (KD) siRNA. Error bars represent SEM, ***=p=0.0003, ns=p=0.7432, n=5.
    Figure Legend Snippet: XRN1 knockdown in SAOS-2 cells does not result in observable phenotypes. A) Successful knockdown of XRN1 in SAOS-2 cells using RNAi 24 hours post transfection. Scr samples treated with 20pmol scrambled siRNA and KD cells treated with 20pmol XRN1 siRNA. Error bars represent SEM, ***=p=0.0008. B) Quantification and representative images (40x objective) of the BrdU proliferation assay. Error bars represent SEM, n≥25, p=0.7938, scale bar=50µM. C) WST-1 assay at 24hr time intervals following transfection with either Scrambled (Scr) or XRN1 (KD) siRNA. Error bar represent SEM, n=3. D) Caspase Glo 3/4 assay at 24hr time intervals following transfection with either Scrambled (Scr) or XRN1 (KD) siRNA. Error bar represent SEM, n=3. E) Quantification and representative images (20x objective) of transwell migration assay 6hrs, 24hrs or 30hrs post seeding. Seeding was performed 24hrs post transfection with either Scrambled (Scr) or XRN1 (KD) siRNA. Error bars represent SEM, n=4, p > 0.05, scale bar=100µM. F) Knockdown of XRN1 does not affect nascent translation rates. Quantification of Puromycin incorporation or XRN1 expression (normalised to GAPDH relative to its own scrambled partner) 24hrs post transfection in cells treated with either Scrambled (Scr) or XRN1 (KD) siRNA. Error bars represent SEM, ***=p=0.0003, ns=p=0.7432, n=5.

    Techniques Used: Transfection, Proliferation Assay, WST-1 Assay, Transwell Migration Assay, Expressing

    3) Product Images from "Global profiling of cellular substrates of human Dcp2"

    Article Title: Global profiling of cellular substrates of human Dcp2

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.0c00069

    Dcp2 targeting of RNAs correlates with P-body enrichment and is enhanced by m 6 A modification. (a-e) Boxplots quantifying differential expression of RNAs after DDX6 silencing in HEK293T cells relative to control (data from Courel et al. , 2019) (a), translation efficiency (data from Sidrauski et al. , 2015) (b), 5′ UTR length (c), 3′ UTR length (reference datasets from Khong et al. , 2017) (d), and RNA enrichment after XRN1 knockout in HEK293T cells (data from Chang et al ., 2019) (e) for each of the three classes of mRNA stability changes in DCP2 KO versus WT HEK293T cells. Statistical significance is calculated using Mann-Whitney U test. Ns, not significant (p > 0.05); ***P
    Figure Legend Snippet: Dcp2 targeting of RNAs correlates with P-body enrichment and is enhanced by m 6 A modification. (a-e) Boxplots quantifying differential expression of RNAs after DDX6 silencing in HEK293T cells relative to control (data from Courel et al. , 2019) (a), translation efficiency (data from Sidrauski et al. , 2015) (b), 5′ UTR length (c), 3′ UTR length (reference datasets from Khong et al. , 2017) (d), and RNA enrichment after XRN1 knockout in HEK293T cells (data from Chang et al ., 2019) (e) for each of the three classes of mRNA stability changes in DCP2 KO versus WT HEK293T cells. Statistical significance is calculated using Mann-Whitney U test. Ns, not significant (p > 0.05); ***P

    Techniques Used: Modification, Expressing, Knock-Out, MANN-WHITNEY

    Validation of transcript stabilization in DCP2 KO cells. (a) The stabilities of selected Dcp2 targets were measured using qRT-PCR in DCP2 KO versus WT HEK293T cells treated with actinomycin D for the indicated times. Half-lives (95% CI) were obtained from linear regression analysis, and significance was determined using two-tailed t -test of the slope of regression lines. Error bars represent mean ± s.d. n =4 biological replicates for HOXA13 , GATA6 and CCNT1 and n in WT, XRN1 KO and XRN1/DCP2 double knockout (DKO) HEK293T cell lines was performed to assay decapping of selected Dcp2 targets. Significance was analyzed by ANOVA linear regression (total RNA) or at individual time points (splint ligation). Error bars represent mean ± s.d. n =4 biological replicates. P-values are denoted by asterisks; *P
    Figure Legend Snippet: Validation of transcript stabilization in DCP2 KO cells. (a) The stabilities of selected Dcp2 targets were measured using qRT-PCR in DCP2 KO versus WT HEK293T cells treated with actinomycin D for the indicated times. Half-lives (95% CI) were obtained from linear regression analysis, and significance was determined using two-tailed t -test of the slope of regression lines. Error bars represent mean ± s.d. n =4 biological replicates for HOXA13 , GATA6 and CCNT1 and n in WT, XRN1 KO and XRN1/DCP2 double knockout (DKO) HEK293T cell lines was performed to assay decapping of selected Dcp2 targets. Significance was analyzed by ANOVA linear regression (total RNA) or at individual time points (splint ligation). Error bars represent mean ± s.d. n =4 biological replicates. P-values are denoted by asterisks; *P

    Techniques Used: Quantitative RT-PCR, Two Tailed Test, Double Knockout, Ligation

    4) Product Images from "CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex"

    Article Title: CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex

    Journal: bioRxiv

    doi: 10.1101/811018

    SMG6-mediated endocleavage is impaired when CASC3 is not present. A : Schematic depiction of the globin mRNA reporter. The reporter consists of three exons (orange boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the second exon. B : Northern blot of RNA extracted from the indicated cell lines that stably express the globin reporter. The xrFrag corresponds to the 3′ part of the reporter that is resistant to degradation by XRN1 due to the xrRNA. The cell lines in lane 3 and 6 were additionally treated with XRN1 siRNA which results in the appearance of a 3′ degradation fragment below the full-length reporter. Reporter and 3′ fragment mRNA levels were normalized to 7SL RNA which is shown as a loading control. For the relative mRNA quantification, in each condition (WT vs. CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the globin WT reporter (lanes 1 and 4). Individual data points and means are plotted from n=3 experiments. C : Schematic depiction of the TOE1 minigene reporter consisting of exons 6-8 (purple boxes) followed by a probe binding cassette (gray boxes). The reporter can be spliced to either contain the canonical stop codon (bottom right) or, by usage of an alternative 3′ splice site, a PTC in exon 7 (top right). D : Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing the TOE1 minigene reporter. The 3′ fragment levels were first normalized to the 7SL RNA loading control and for every cell line the XRN1 knockdown condition to the condition without XRN1 knockdown (n=2). E : Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing a prespliced variant of the TOE1 minigene reporter depicted in Figure 5C. The intron between exon 6 and 7 is deleted so that the reporter is constitutively spliced to contain either a normal stop codon (TOE1-WT) or a PTC (TOE1-PTC). The mRNA levels were normalized to 7SL RNA. For the relative mRNA quantification, in each condition (WT vs. CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the TOE-WT reporter (lanes 1 and 4). F : Schematic depiction of the triose phosphate isomerase (TPI) mRNA reporter. The reporter consists of seven exons (blue boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the fifth exon. G : Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing the either the TPI WT or TPI PTC mRNA reporter. The reporter and 3′ fragment mRNA levels were normalized to the 7SL control. For each cell line, the mRNA levels were then normalized to the respective TPI WT reporter or 3′ fragment levels. Individual data points and means are plotted from n=3 experiments.
    Figure Legend Snippet: SMG6-mediated endocleavage is impaired when CASC3 is not present. A : Schematic depiction of the globin mRNA reporter. The reporter consists of three exons (orange boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the second exon. B : Northern blot of RNA extracted from the indicated cell lines that stably express the globin reporter. The xrFrag corresponds to the 3′ part of the reporter that is resistant to degradation by XRN1 due to the xrRNA. The cell lines in lane 3 and 6 were additionally treated with XRN1 siRNA which results in the appearance of a 3′ degradation fragment below the full-length reporter. Reporter and 3′ fragment mRNA levels were normalized to 7SL RNA which is shown as a loading control. For the relative mRNA quantification, in each condition (WT vs. CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the globin WT reporter (lanes 1 and 4). Individual data points and means are plotted from n=3 experiments. C : Schematic depiction of the TOE1 minigene reporter consisting of exons 6-8 (purple boxes) followed by a probe binding cassette (gray boxes). The reporter can be spliced to either contain the canonical stop codon (bottom right) or, by usage of an alternative 3′ splice site, a PTC in exon 7 (top right). D : Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing the TOE1 minigene reporter. The 3′ fragment levels were first normalized to the 7SL RNA loading control and for every cell line the XRN1 knockdown condition to the condition without XRN1 knockdown (n=2). E : Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing a prespliced variant of the TOE1 minigene reporter depicted in Figure 5C. The intron between exon 6 and 7 is deleted so that the reporter is constitutively spliced to contain either a normal stop codon (TOE1-WT) or a PTC (TOE1-PTC). The mRNA levels were normalized to 7SL RNA. For the relative mRNA quantification, in each condition (WT vs. CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the TOE-WT reporter (lanes 1 and 4). F : Schematic depiction of the triose phosphate isomerase (TPI) mRNA reporter. The reporter consists of seven exons (blue boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the fifth exon. G : Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing the either the TPI WT or TPI PTC mRNA reporter. The reporter and 3′ fragment mRNA levels were normalized to the 7SL control. For each cell line, the mRNA levels were then normalized to the respective TPI WT reporter or 3′ fragment levels. Individual data points and means are plotted from n=3 experiments.

    Techniques Used: Binding Assay, Northern Blot, Stable Transfection, Expressing, Variant Assay

    The CASC3 N-terminus promotes but is not necessary to elicit NMD. A : Schematic depiction of the TPI-MS2V5-SMG5 tethering reporter. The reporter consists of the TPI ORF (blue boxes) followed by 4 MS2 stem loops (SL). Downstream the SMG5 3′ untranslated region (UTR) is inserted to increase the size of 3′ fragments that result from cleavage at the termination codon. Reporter and 3′ fragment mRNAs can be detected via the probe binding cassette (gray boxes). B : Northern blot of a tethering assay performed in HeLa Tet-Off cells. The cells stably express the tethering reporter shown in Figure 6A together with the indicated MS2V5-tagged proteins. When the cells are additionally treated with XRN1 siRNA, a 3′ degradation fragment can be detected below the full-length reporter. The reporter and 3′ fragment mRNA levels are normalized to the 7SL RNA. For the calculation of the relative mRNA levels in each condition (Luc vs. XRN1) the levels were normalized to the MS2V5-GST control (lanes 1 and 5). C : Schematic depiction of CASC3 rescue protein constructs. The full-length (FL) protein consists of an N-terminal (blue), C-terminal (orange) and central SELOR domain (purple). The construct 1-480 has a C-terminal deletion, whereas in the construct 110-480 both the N- and C-terminus are truncated. Both deletion constructs were also rendered EJC-binding deficient by mutating the amino acid residues 188 and 218 (F188D, W218D). D : Relative quantification of the CLN6 (top) and TOE1 (bottom) transcript isoforms by qPCR in the indicated cell lines. The V5-tagged rescue proteins expressed in the KO condition are shown schematically in Figure 6C. Rescue protein expression is confirmed in Figure 6E and F. Individual data points and means are plotted from n=3 experiments. E and F: Western blot of samples shown in Figure 6D. The expression of rescue proteins was confirmed by an antibody against CASC3 (E) and an antibody recognizing the V5 tag (F).
    Figure Legend Snippet: The CASC3 N-terminus promotes but is not necessary to elicit NMD. A : Schematic depiction of the TPI-MS2V5-SMG5 tethering reporter. The reporter consists of the TPI ORF (blue boxes) followed by 4 MS2 stem loops (SL). Downstream the SMG5 3′ untranslated region (UTR) is inserted to increase the size of 3′ fragments that result from cleavage at the termination codon. Reporter and 3′ fragment mRNAs can be detected via the probe binding cassette (gray boxes). B : Northern blot of a tethering assay performed in HeLa Tet-Off cells. The cells stably express the tethering reporter shown in Figure 6A together with the indicated MS2V5-tagged proteins. When the cells are additionally treated with XRN1 siRNA, a 3′ degradation fragment can be detected below the full-length reporter. The reporter and 3′ fragment mRNA levels are normalized to the 7SL RNA. For the calculation of the relative mRNA levels in each condition (Luc vs. XRN1) the levels were normalized to the MS2V5-GST control (lanes 1 and 5). C : Schematic depiction of CASC3 rescue protein constructs. The full-length (FL) protein consists of an N-terminal (blue), C-terminal (orange) and central SELOR domain (purple). The construct 1-480 has a C-terminal deletion, whereas in the construct 110-480 both the N- and C-terminus are truncated. Both deletion constructs were also rendered EJC-binding deficient by mutating the amino acid residues 188 and 218 (F188D, W218D). D : Relative quantification of the CLN6 (top) and TOE1 (bottom) transcript isoforms by qPCR in the indicated cell lines. The V5-tagged rescue proteins expressed in the KO condition are shown schematically in Figure 6C. Rescue protein expression is confirmed in Figure 6E and F. Individual data points and means are plotted from n=3 experiments. E and F: Western blot of samples shown in Figure 6D. The expression of rescue proteins was confirmed by an antibody against CASC3 (E) and an antibody recognizing the V5 tag (F).

    Techniques Used: Binding Assay, Northern Blot, Stable Transfection, Construct, Real-time Polymerase Chain Reaction, Expressing, Western Blot

    5) Product Images from "RNA triphosphatase DUSP11 enables exonuclease XRN-mediated restriction of hepatitis C virus"

    Article Title: RNA triphosphatase DUSP11 enables exonuclease XRN-mediated restriction of hepatitis C virus

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1802326115

    HCV restriction activity of XRNs is reduced in cells lacking DUSP11. ( A ) sgJFH1-Rluc-GND (nonreplicative polymerase mutant) replicon luciferase assay in parental Huh7 cells and two independent DUSP11 knockout clones (D11-KO-8 and D11-KO-9). Luciferase assays were performed at the indicated times post replicon RNA transfection. The mean ± SEM of three individual experiments is presented. ( B ) Confirmation of siRNA knockdown of XRN1 and XRN2 in parental Huh7 cells and two independent DUSP11 knockout clones (D11-KO-8 and D11-KO-9) by immunoblot. Huh7 were either mock transfected (Mock) or transfected with either an irrelevant control siRNA or a pool of XRN1- and XRN2-specific siRNAs (siXRNs). Cell lysates were harvested 48 h post transfection and assayed by immunoblot with the indicated antibodies. ( C ) sgJFH1-Rluc replicon luciferase assay in parental Huh7 cells and two independent DUSP11 knockout clones (D11-KO-8 and D11-KO-9) treated with siNC or a pool of siXRNs. Luciferase assays were performed at the indicated times post replicon RNA transfection. The mean ± SEM of three experiments is presented. ( D ) Huh7 and D11-KO-9 cells were infected with HCV for 96 h and treated with 5 μM sofosbuvir, and HCV RNA levels were quantified at the indicated times post treatment. The mean ± SEM of eight samples is presented. Statistical significance was assessed by Student’s t test and is indicated as follows: * P
    Figure Legend Snippet: HCV restriction activity of XRNs is reduced in cells lacking DUSP11. ( A ) sgJFH1-Rluc-GND (nonreplicative polymerase mutant) replicon luciferase assay in parental Huh7 cells and two independent DUSP11 knockout clones (D11-KO-8 and D11-KO-9). Luciferase assays were performed at the indicated times post replicon RNA transfection. The mean ± SEM of three individual experiments is presented. ( B ) Confirmation of siRNA knockdown of XRN1 and XRN2 in parental Huh7 cells and two independent DUSP11 knockout clones (D11-KO-8 and D11-KO-9) by immunoblot. Huh7 were either mock transfected (Mock) or transfected with either an irrelevant control siRNA or a pool of XRN1- and XRN2-specific siRNAs (siXRNs). Cell lysates were harvested 48 h post transfection and assayed by immunoblot with the indicated antibodies. ( C ) sgJFH1-Rluc replicon luciferase assay in parental Huh7 cells and two independent DUSP11 knockout clones (D11-KO-8 and D11-KO-9) treated with siNC or a pool of siXRNs. Luciferase assays were performed at the indicated times post replicon RNA transfection. The mean ± SEM of three experiments is presented. ( D ) Huh7 and D11-KO-9 cells were infected with HCV for 96 h and treated with 5 μM sofosbuvir, and HCV RNA levels were quantified at the indicated times post treatment. The mean ± SEM of eight samples is presented. Statistical significance was assessed by Student’s t test and is indicated as follows: * P

    Techniques Used: Activity Assay, Mutagenesis, Luciferase, Knock-Out, Clone Assay, Transfection, Infection

    DUSP11 directly dephosphorylates HCV 5′ UTR RNA and sensitizes it to XRN-mediated degradation. ( A ) Confirmation of in vitro translation products by immunoblot. Membrane was incubated with the indicated antibodies. “pLuciferase” indicates reactions programmed to express luciferase as a negative control. ( B ) In vitro phosphatase assay. 5′ γ- 32 P–radiolabeled HCV 5′ UTR RNA was incubated with the indicated enzymes (calf intestinal phosphatase, purified DUSP11 core protein, and bacterial 5′ RNA polyphosphatase), or in vitro translated products from A (pDUSP11, pDUSP11-CM, and negative-control pLuciferase). Products were separated by urea/PAGE and stained with ethidium bromide. Products were then transferred to a nitrocellulose membrane and exposed to a phosphor storage screen (Phos). ( C ) In vitro XRN susceptibility assay. In vitro phosphatase reactions were performed as in B [calf intestinal phosphatase, bacterial 5′ RNA polyphosphatase, or in vitro translated products from A (pDUSP11, pDUSP11-CM, and negative-control pLuciferase)], but products were recovered and incubated ± recombinant XRN1. Products were separated by urea/PAGE and stained with EtBr. “FL” arrow points to the position of the full-length HCV 5′ UTR. “D” arrow points to the position of a faster-migrating degradation product.
    Figure Legend Snippet: DUSP11 directly dephosphorylates HCV 5′ UTR RNA and sensitizes it to XRN-mediated degradation. ( A ) Confirmation of in vitro translation products by immunoblot. Membrane was incubated with the indicated antibodies. “pLuciferase” indicates reactions programmed to express luciferase as a negative control. ( B ) In vitro phosphatase assay. 5′ γ- 32 P–radiolabeled HCV 5′ UTR RNA was incubated with the indicated enzymes (calf intestinal phosphatase, purified DUSP11 core protein, and bacterial 5′ RNA polyphosphatase), or in vitro translated products from A (pDUSP11, pDUSP11-CM, and negative-control pLuciferase). Products were separated by urea/PAGE and stained with ethidium bromide. Products were then transferred to a nitrocellulose membrane and exposed to a phosphor storage screen (Phos). ( C ) In vitro XRN susceptibility assay. In vitro phosphatase reactions were performed as in B [calf intestinal phosphatase, bacterial 5′ RNA polyphosphatase, or in vitro translated products from A (pDUSP11, pDUSP11-CM, and negative-control pLuciferase)], but products were recovered and incubated ± recombinant XRN1. Products were separated by urea/PAGE and stained with EtBr. “FL” arrow points to the position of the full-length HCV 5′ UTR. “D” arrow points to the position of a faster-migrating degradation product.

    Techniques Used: In Vitro, Incubation, Luciferase, Negative Control, Phosphatase Assay, Purification, Polyacrylamide Gel Electrophoresis, Staining, Drug Susceptibility Assay, Recombinant

    6) Product Images from "The m6A reader protein YTHDC2 interacts with the small ribosomal subunit and the 5′–3′ exoribonuclease XRN1"

    Article Title: The m6A reader protein YTHDC2 interacts with the small ribosomal subunit and the 5′–3′ exoribonuclease XRN1

    Journal: RNA

    doi: 10.1261/rna.064238.117

    Both the YTH and R3H domains contribute to RNA binding by YTHDC2 and YTHDC2 interacts specifically with the 5′–3′ exoribonuclease XRN1 via its ANK repeats. ( A ) Cells expressing equal amounts of Flag-tagged versions of full-length YTHDC2, YTHDC2 lacking the R3H domain (ΔR3H), and YTHDC2 lacking the YTH domain (ΔYTH) were treated with 4-thiouridine and crosslinked in vivo. After tandem affinity purification of crosslinked protein–RNA complexes, RNA trimming and 5′ labeling with [ 32 P], complexes were separated by denaturing PAGE, transferred to a nitrocellulose membrane, and radioactively labeled RNAs were detected using autoradiography. Protein eluates were analyzed by western blotting using an anti-Flag antibody. ( B ) Extracts from HEK293 cells expressing Flag-tagged YTHDC2, YTHDC1, YTHDF1, YTHDF2, YTHDF3, or the Flag tag were used in immunoprecipitation experiments in the presence (+) or absence (−) of RNase A and T1 (RNase). Inputs (1%) and eluates (IP) were analyzed by western blotting using antibodies against XRN1, GAPDH, and the Flag tag. ( C ) Immunoprecipitation experiments were performed and analyzed as in B in the absence of RNase treatment using extracts prepared from cells expressing Flag-tagged full-length YTHDC2 (YTHDC2), YTHDC2 lacking the R3H domain (ΔR3H), the YTH domain (ΔYTH), or one or both ankyrin repeats (ΔANK1, ΔANK2, ΔANK1+2). All experiments presented in this figure were performed in duplicate or triplicate and representative data are shown.
    Figure Legend Snippet: Both the YTH and R3H domains contribute to RNA binding by YTHDC2 and YTHDC2 interacts specifically with the 5′–3′ exoribonuclease XRN1 via its ANK repeats. ( A ) Cells expressing equal amounts of Flag-tagged versions of full-length YTHDC2, YTHDC2 lacking the R3H domain (ΔR3H), and YTHDC2 lacking the YTH domain (ΔYTH) were treated with 4-thiouridine and crosslinked in vivo. After tandem affinity purification of crosslinked protein–RNA complexes, RNA trimming and 5′ labeling with [ 32 P], complexes were separated by denaturing PAGE, transferred to a nitrocellulose membrane, and radioactively labeled RNAs were detected using autoradiography. Protein eluates were analyzed by western blotting using an anti-Flag antibody. ( B ) Extracts from HEK293 cells expressing Flag-tagged YTHDC2, YTHDC1, YTHDF1, YTHDF2, YTHDF3, or the Flag tag were used in immunoprecipitation experiments in the presence (+) or absence (−) of RNase A and T1 (RNase). Inputs (1%) and eluates (IP) were analyzed by western blotting using antibodies against XRN1, GAPDH, and the Flag tag. ( C ) Immunoprecipitation experiments were performed and analyzed as in B in the absence of RNase treatment using extracts prepared from cells expressing Flag-tagged full-length YTHDC2 (YTHDC2), YTHDC2 lacking the R3H domain (ΔR3H), the YTH domain (ΔYTH), or one or both ankyrin repeats (ΔANK1, ΔANK2, ΔANK1+2). All experiments presented in this figure were performed in duplicate or triplicate and representative data are shown.

    Techniques Used: RNA Binding Assay, Expressing, In Vivo, Affinity Purification, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Western Blot, FLAG-tag, Immunoprecipitation

    7) Product Images from "Interrogating the degradation pathways of unstable mRNAs with XRN1-resistant sequences"

    Article Title: Interrogating the degradation pathways of unstable mRNAs with XRN1-resistant sequences

    Journal: Nature Communications

    doi: 10.1038/ncomms13691

    Characterization of xrRNA elements enabling the detection of mRNA degradation intermediates. ( a ) Depicted are the general mRNA degradation pathways leading either directly (decapping and endocleavage) or indirectly (deadenylation) to 5′–3′ decay executed by XRN1. The presence of a stable XRN1-resistant RNA structure (xrRNA) prevents XRN1 from further progression and thus protects the remaining RNA fragment (xrFrag) from degradation from the 5′ end. ( b ) The DNA sequence of the xrRNA element used in reporter constructs is shown with annotations of sequence motifs. ( c , e ) Schematic representation of the TPI reporter mRNA. The TPI gene is depicted as blue boxes representing single exons (exon numbers indicated). The positions of the normal stop codons (stop) and premature translation termination codons are shown. Northern blot probe binding sites in the 3′ UTR are depicted as grey boxes and single xrRNA structures 1 and 2 are shown in red and purple. The difference between full xrRNA and 1+2 is the presence or absence of a short spacer region (indicated in b ). ( e ) The 60 bp elements with varying GC content were derived from the RAB7A 3′ UTR, the 4MS2 binding sites are identical to those used in tethering experiments ( Fig. 5 ) and the stem–loop structure is used in other reporters to block translation initiation ( Fig. 5 ). ( d , f ) Northern blots of RNA samples extracted from HeLa cells transfected with the indicated reporter constructs. Co-transfected LacZ served as control mRNA. ( f ) Mean values of reporter and xrFrag signal±s.d. ( n =3) were quantified and normalized to the TPI reporter without insert. The ratio of xrFrag to reporter mRNA levels is indicated below the graph.
    Figure Legend Snippet: Characterization of xrRNA elements enabling the detection of mRNA degradation intermediates. ( a ) Depicted are the general mRNA degradation pathways leading either directly (decapping and endocleavage) or indirectly (deadenylation) to 5′–3′ decay executed by XRN1. The presence of a stable XRN1-resistant RNA structure (xrRNA) prevents XRN1 from further progression and thus protects the remaining RNA fragment (xrFrag) from degradation from the 5′ end. ( b ) The DNA sequence of the xrRNA element used in reporter constructs is shown with annotations of sequence motifs. ( c , e ) Schematic representation of the TPI reporter mRNA. The TPI gene is depicted as blue boxes representing single exons (exon numbers indicated). The positions of the normal stop codons (stop) and premature translation termination codons are shown. Northern blot probe binding sites in the 3′ UTR are depicted as grey boxes and single xrRNA structures 1 and 2 are shown in red and purple. The difference between full xrRNA and 1+2 is the presence or absence of a short spacer region (indicated in b ). ( e ) The 60 bp elements with varying GC content were derived from the RAB7A 3′ UTR, the 4MS2 binding sites are identical to those used in tethering experiments ( Fig. 5 ) and the stem–loop structure is used in other reporters to block translation initiation ( Fig. 5 ). ( d , f ) Northern blots of RNA samples extracted from HeLa cells transfected with the indicated reporter constructs. Co-transfected LacZ served as control mRNA. ( f ) Mean values of reporter and xrFrag signal±s.d. ( n =3) were quantified and normalized to the TPI reporter without insert. The ratio of xrFrag to reporter mRNA levels is indicated below the graph.

    Techniques Used: Sequencing, Construct, Northern Blot, Binding Assay, Derivative Assay, Blocking Assay, Transfection

    8) Product Images from "Transcript-specific characteristics determine the contribution of endo- and exonucleolytic decay pathways during the degradation of nonsense-mediated decay substrates"

    Article Title: Transcript-specific characteristics determine the contribution of endo- and exonucleolytic decay pathways during the degradation of nonsense-mediated decay substrates

    Journal: RNA

    doi: 10.1261/rna.059659.116

    Deletions of C-terminal UPF1 phosphorylation sites impair NMD differently. ( A ) UPF1 protein architecture is depicted schematically. All structural and functional domains are indicated; the presence of potential phosphorylation sites (SQ/TQ) are shown in red and blue, respectively. ( B , C ) Northern blots of RNA samples extracted from HeLa cells transfected with the indicated siRNAs and reporter constructs. Cotransfected LacZ-H4 ( B ) or β-globin ( C ) served as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. Representative Western blots are shown at the bottom , using tubulin as loading control.
    Figure Legend Snippet: Deletions of C-terminal UPF1 phosphorylation sites impair NMD differently. ( A ) UPF1 protein architecture is depicted schematically. All structural and functional domains are indicated; the presence of potential phosphorylation sites (SQ/TQ) are shown in red and blue, respectively. ( B , C ) Northern blots of RNA samples extracted from HeLa cells transfected with the indicated siRNAs and reporter constructs. Cotransfected LacZ-H4 ( B ) or β-globin ( C ) served as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. Representative Western blots are shown at the bottom , using tubulin as loading control.

    Techniques Used: Functional Assay, Northern Blot, Transfection, Construct, Western Blot

    Functional analysis of potential endogenous NMD targets identified during high-throughput sequencing. ( A – D ) A PCR-based approach was used to quantify 3′ fragment levels upon knockdown of NMD effectors. HeLa cells were transiently transfected with the indicated siRNAs and poly(A) + RNA was extracted. 3′ Decay intermediates were ligated to an RNA linker, followed by reverse transcription with an oligo(dT) primer and gene-specific PCR. Overall transcript levels were determined with primer pairs located downstream from the estimated endocleavage site (second panel each, indicated as “gene int.”). PCR with TATA-Box binding protein (TBP) primers was used for cDNA level determination. For each class of NMD targets ( A , B , C , D, respectively), the same set of cDNA was used and therefore the same TBP profiles are shown for each target type. Degradome-sequencing reads were plotted against their position on the indicated mRNAs. Endocleavage events within the selected targets were visualized in an enlarged view spanning 150 nt (±75 nt). The position of the second nucleotide of the respective stop codon is set as zero. Mapped reads per nucleotide were plotted against the mRNA length for each knockdown condition (Luciferase [Luc, black], XRN1 [orange], XRN1/SMG6 [blue]). ( A ) PDRG1, SURF6, and MED10 transcripts with a long 3′ UTR. ( B ) IFRD1 and BAG1 transcripts with an uORF. ( C ) C11orf31, encoding for Selenoprotein H, containing a selenocysteine (Sec) codon. ( D ) TMEM222, incorporation of an alternative exon (indicated in purple), harboring a PTC as well as RPL10A, a transcript gaining a PTC probably due to alternative splice site usage within the third intron.
    Figure Legend Snippet: Functional analysis of potential endogenous NMD targets identified during high-throughput sequencing. ( A – D ) A PCR-based approach was used to quantify 3′ fragment levels upon knockdown of NMD effectors. HeLa cells were transiently transfected with the indicated siRNAs and poly(A) + RNA was extracted. 3′ Decay intermediates were ligated to an RNA linker, followed by reverse transcription with an oligo(dT) primer and gene-specific PCR. Overall transcript levels were determined with primer pairs located downstream from the estimated endocleavage site (second panel each, indicated as “gene int.”). PCR with TATA-Box binding protein (TBP) primers was used for cDNA level determination. For each class of NMD targets ( A , B , C , D, respectively), the same set of cDNA was used and therefore the same TBP profiles are shown for each target type. Degradome-sequencing reads were plotted against their position on the indicated mRNAs. Endocleavage events within the selected targets were visualized in an enlarged view spanning 150 nt (±75 nt). The position of the second nucleotide of the respective stop codon is set as zero. Mapped reads per nucleotide were plotted against the mRNA length for each knockdown condition (Luciferase [Luc, black], XRN1 [orange], XRN1/SMG6 [blue]). ( A ) PDRG1, SURF6, and MED10 transcripts with a long 3′ UTR. ( B ) IFRD1 and BAG1 transcripts with an uORF. ( C ) C11orf31, encoding for Selenoprotein H, containing a selenocysteine (Sec) codon. ( D ) TMEM222, incorporation of an alternative exon (indicated in purple), harboring a PTC as well as RPL10A, a transcript gaining a PTC probably due to alternative splice site usage within the third intron.

    Techniques Used: Functional Assay, Next-Generation Sequencing, Polymerase Chain Reaction, Transfection, Binding Assay, Sequencing, Luciferase, Size-exclusion Chromatography

    SMG7 abundance influences endocleavage efficiency for long 3′ UTR targets. ( A ) Domain structure of SMG7, showing the N-terminal 14-3-3-like domain (interacts with UPF1) and α-helical extensions as well as the C-terminal PC region (interacts with POP2). ( B ) Northern blot of RNA samples extracted from HeLa cells transfected with the indicated siRNAs and reporter constructs. Cotransfected β-globin served as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. A representative Western blot is shown at the bottom , using tubulin as loading control. ( C ) Schematic representation of the transfected triosephosphate isomerase (TPI) reporter with a PTC at amino acid position 160. Exons are depicted as white (untranslated) or black (translated) boxes, introns as two connecting black lines, and the Northern probe binding sites as light gray boxes. Vector derived 5′ UTR intron and SV40 poly(A) signal (pA) are shown. ( D ) Northern blot of RNA samples extracted from HeLa cells transfected with the indicated siRNAs, plasmids, and reporter constructs using LacZ-4H as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. A representative Western blot is shown at the bottom , using tubulin as loading control.
    Figure Legend Snippet: SMG7 abundance influences endocleavage efficiency for long 3′ UTR targets. ( A ) Domain structure of SMG7, showing the N-terminal 14-3-3-like domain (interacts with UPF1) and α-helical extensions as well as the C-terminal PC region (interacts with POP2). ( B ) Northern blot of RNA samples extracted from HeLa cells transfected with the indicated siRNAs and reporter constructs. Cotransfected β-globin served as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. A representative Western blot is shown at the bottom , using tubulin as loading control. ( C ) Schematic representation of the transfected triosephosphate isomerase (TPI) reporter with a PTC at amino acid position 160. Exons are depicted as white (untranslated) or black (translated) boxes, introns as two connecting black lines, and the Northern probe binding sites as light gray boxes. Vector derived 5′ UTR intron and SV40 poly(A) signal (pA) are shown. ( D ) Northern blot of RNA samples extracted from HeLa cells transfected with the indicated siRNAs, plasmids, and reporter constructs using LacZ-4H as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. A representative Western blot is shown at the bottom , using tubulin as loading control.

    Techniques Used: Northern Blot, Transfection, Construct, Western Blot, Binding Assay, Plasmid Preparation, Derivative Assay

    9) Product Images from "Cellular 5′-3′ mRNA Exonuclease Xrn1 Controls Double-stranded RNA Accumulation and Anti-Viral Responses"

    Article Title: Cellular 5′-3′ mRNA Exonuclease Xrn1 Controls Double-stranded RNA Accumulation and Anti-Viral Responses

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2015.02.003

    Massive dsRNA accumulation in Xrn1-depleted cells infected with VacV NHDFs transfected with the indicated siRNAs were mock-infected or infected with VacV (MOI = 5). Cells were fixed at 6, 12, and 18 hpi and stained for immunofluorescence with J2 anti-dsRNA antibody ( green ). DNA was stained using DAPI ( blue ). (A) Cells were visualized using fluorescence microscopy with a 20X objective. (B) NHDFs treated with siRNAs and infected as in (A) were harvested and cell free lysates prepared at 18 hpi. Equal volumes of lysates were dotted onto membrane and dsRNA detected by Immunoblotting ( upper panel ). The dsRNA signal from B together with two independent replicates was quantified and the means plotted ± SEM ( lower panel ). (C) Confocal image of Xrn1 siRNA-treated, infected cells from A, fixed at 18 hpi using 63X objective. (D) Xrn1 siRNA-treated, infected cells fixed at 18 hpi were treated with a mixture of single strand-specific RNase A/T1, dsRNA-specific RNase III or buffer alone prior to immunostaining of dsRNA.
    Figure Legend Snippet: Massive dsRNA accumulation in Xrn1-depleted cells infected with VacV NHDFs transfected with the indicated siRNAs were mock-infected or infected with VacV (MOI = 5). Cells were fixed at 6, 12, and 18 hpi and stained for immunofluorescence with J2 anti-dsRNA antibody ( green ). DNA was stained using DAPI ( blue ). (A) Cells were visualized using fluorescence microscopy with a 20X objective. (B) NHDFs treated with siRNAs and infected as in (A) were harvested and cell free lysates prepared at 18 hpi. Equal volumes of lysates were dotted onto membrane and dsRNA detected by Immunoblotting ( upper panel ). The dsRNA signal from B together with two independent replicates was quantified and the means plotted ± SEM ( lower panel ). (C) Confocal image of Xrn1 siRNA-treated, infected cells from A, fixed at 18 hpi using 63X objective. (D) Xrn1 siRNA-treated, infected cells fixed at 18 hpi were treated with a mixture of single strand-specific RNase A/T1, dsRNA-specific RNase III or buffer alone prior to immunostaining of dsRNA.

    Techniques Used: Infection, Transfection, Staining, Immunofluorescence, Fluorescence, Microscopy, Immunostaining

    Increased sensitivity to the dsRNA analog poly(I:C) in response to Xrn1-depletion in uninfected cells NHDFs treated with the indicated siRNAs were mock-transfected or transfected with increasing amounts of poly(I:C). After 3h, cells were metabolically pulse-labeled with [ 35 S]Met-Cys for 30 min. Total protein was collected, separated by SDS-PAGE, and 35 .
    Figure Legend Snippet: Increased sensitivity to the dsRNA analog poly(I:C) in response to Xrn1-depletion in uninfected cells NHDFs treated with the indicated siRNAs were mock-transfected or transfected with increasing amounts of poly(I:C). After 3h, cells were metabolically pulse-labeled with [ 35 S]Met-Cys for 30 min. Total protein was collected, separated by SDS-PAGE, and 35 .

    Techniques Used: Transfection, Metabolic Labelling, Labeling, SDS Page

    Phosphorylated eIF2α accumulation in Xrn1-depleted cells correlates with global protein synthesis inhibition late in the VacV lifecycle NHDFs transfected with ns control or Xrn1-specific siRNAs were infected with VacV (MOI = 5). At the indicated times (hpi) cells were metabolically pulse-labeled with [ 35 S]Met-Cys for 30 min. Uninfected cells (UI) were harvested in parallel with 18 hpi samples. (A) Total protein was isolated, separated by SDS-PAGE and the fixed, dried gel exposed to X-ray film. Molecular mass standards (in kDa) are shown to the right. (B) The same lysates were immunoblotted with the indicated antibodies. Tubulin served as a loading control. (C) RNA from NHDFs treated with the indicated siRNAs and infected as in A was harvested at 6 hpi and subject to RT-qPCR using primers specific for K3L, E3L or Xrn1 mRNAs. Each reaction product was normalized to the signal obtained using primers specific for 18S rRNA and expressed as the fold change relative to control siRNA-treated cells. Means of 3 independent experiments are plotted ± SEM.
    Figure Legend Snippet: Phosphorylated eIF2α accumulation in Xrn1-depleted cells correlates with global protein synthesis inhibition late in the VacV lifecycle NHDFs transfected with ns control or Xrn1-specific siRNAs were infected with VacV (MOI = 5). At the indicated times (hpi) cells were metabolically pulse-labeled with [ 35 S]Met-Cys for 30 min. Uninfected cells (UI) were harvested in parallel with 18 hpi samples. (A) Total protein was isolated, separated by SDS-PAGE and the fixed, dried gel exposed to X-ray film. Molecular mass standards (in kDa) are shown to the right. (B) The same lysates were immunoblotted with the indicated antibodies. Tubulin served as a loading control. (C) RNA from NHDFs treated with the indicated siRNAs and infected as in A was harvested at 6 hpi and subject to RT-qPCR using primers specific for K3L, E3L or Xrn1 mRNAs. Each reaction product was normalized to the signal obtained using primers specific for 18S rRNA and expressed as the fold change relative to control siRNA-treated cells. Means of 3 independent experiments are plotted ± SEM.

    Techniques Used: Inhibition, Transfection, Infection, Metabolic Labelling, Labeling, Isolation, SDS Page, Quantitative RT-PCR

    Inhibition of protein synthesis following Xrn1-depletion requires a VacV specific late gene transcription factor (A) NHDFs transfected with ns control or Xrn1-specific siRNAs (−1 and −2) were mock-infected or infected with VacV (MOI = 5) in the presence or absence of PAA. At 18 hpi, cells were metabolically pulse-labeled with [ 35 S]Met-Cys for 30 min. Total protein was collected, separated by SDS-PAGE and 35 S -labeled proteins visualized by exposing the fixed, dried gel to X-ray film. Molecular mass standards (in kDa) are shown to the right. (B) The same lysates were also immunoblotted with the indicated antibodies. Tubulin was used as a loading control. VacV I3 (early expressed) serves as an infection control. (C) As in A except NHDFs were infected with WT VacV or an A23-deficient virus (ΔA23). (D) Lysates from C were immunoblotted with the indicated antibodies. eIF2α was used as a loading control.
    Figure Legend Snippet: Inhibition of protein synthesis following Xrn1-depletion requires a VacV specific late gene transcription factor (A) NHDFs transfected with ns control or Xrn1-specific siRNAs (−1 and −2) were mock-infected or infected with VacV (MOI = 5) in the presence or absence of PAA. At 18 hpi, cells were metabolically pulse-labeled with [ 35 S]Met-Cys for 30 min. Total protein was collected, separated by SDS-PAGE and 35 S -labeled proteins visualized by exposing the fixed, dried gel to X-ray film. Molecular mass standards (in kDa) are shown to the right. (B) The same lysates were also immunoblotted with the indicated antibodies. Tubulin was used as a loading control. VacV I3 (early expressed) serves as an infection control. (C) As in A except NHDFs were infected with WT VacV or an A23-deficient virus (ΔA23). (D) Lysates from C were immunoblotted with the indicated antibodies. eIF2α was used as a loading control.

    Techniques Used: Inhibition, Transfection, Infection, Metabolic Labelling, Labeling, SDS Page

    PKR-dependent eIF2α phosphorylation and RNase L-mediated rRNA degradation in Xrn1-depleted cells infected with VacV (A) NHDFs transfected with the indicated siRNAs were mock-infected or infected with VacV (MOI =5). Total protein was collected at 18 hpi and analyzed by immunoblotting with the indicated antibodies. Tubulin served as a loading control. (B) NHDFs transfected with the indicated siRNAs were mock-infected or infected with VacV (MOI =5). At 18 hpi, total RNA was isolated and analyzed using a Bioanalyzer Nano LabChip. 28S and 18S rRNA bands are indicated. (C) NHDFs transfected with the indicated siRNAs were infected as in (A). At 18 hpi, cells were metabolically pulse-labeled with [ 35 S]Met-Cys for 30 min. Total protein was collected, separated by SDS-PAGE and 35 S -labeled proteins visualized by exposing the fixed, dried gel to X-ray film. Molecular mass standards (in KDa) are shown to the right ( upper panel) . The same lysates were also immunoblotted (IB) with the indicated antibodies ( lower panel ). The RNase L-specific immunoreactive band is indicated by an arrow. Tubulin served as a loading control. (D) Metabolically radiolabelled samples from C together with two additional independent replicate experiments were TCA precipitated. 35
    Figure Legend Snippet: PKR-dependent eIF2α phosphorylation and RNase L-mediated rRNA degradation in Xrn1-depleted cells infected with VacV (A) NHDFs transfected with the indicated siRNAs were mock-infected or infected with VacV (MOI =5). Total protein was collected at 18 hpi and analyzed by immunoblotting with the indicated antibodies. Tubulin served as a loading control. (B) NHDFs transfected with the indicated siRNAs were mock-infected or infected with VacV (MOI =5). At 18 hpi, total RNA was isolated and analyzed using a Bioanalyzer Nano LabChip. 28S and 18S rRNA bands are indicated. (C) NHDFs transfected with the indicated siRNAs were infected as in (A). At 18 hpi, cells were metabolically pulse-labeled with [ 35 S]Met-Cys for 30 min. Total protein was collected, separated by SDS-PAGE and 35 S -labeled proteins visualized by exposing the fixed, dried gel to X-ray film. Molecular mass standards (in KDa) are shown to the right ( upper panel) . The same lysates were also immunoblotted (IB) with the indicated antibodies ( lower panel ). The RNase L-specific immunoreactive band is indicated by an arrow. Tubulin served as a loading control. (D) Metabolically radiolabelled samples from C together with two additional independent replicate experiments were TCA precipitated. 35

    Techniques Used: Infection, Transfection, Isolation, Metabolic Labelling, Labeling, SDS Page

    Elevated viral mRNA abundance and their enrichment in dsRNA isolated from Xrn1-depleted, VacV- infected cells Cell free lysates from NHDFs transfected with Xrn1-1 siRNA and infected with VacV (MOI=5) were prepared at 22 hpi and immunoprecipitated using J2 anti-dsRNA antibody. After treating with RNase A/T1 or RNase III, isolated RNA was analyzed by RT-qPCR using the indicated viral or cellular mRNA primers. mRNA abundances were normalized to actin and calculated relative to input (set to 1). The means of three independent experiments are plotted ± SEM. (B) Equal volumes of buffer, input lysate (IP input), or the unbound fraction (IP unbound) were dotted onto a membrane and dsRNA detected by immunoblotting to demonstrate dsRNA depletion in the unbound fraction. (C, D) NHDFs were treated with the indicated siRNAs and RNA isolated from uninfected cells (C) or 3 hpi with VACV (MOI = 5) (D). RNA was subject to RT-qPCR analysis for the indicated cellular or early viral mRNAs and each reaction product normalized to 18S rRNA and presented as the fold change relative to control siRNA-treated cells. The means of 3 independent experiments are plotted ± SEM. A significant difference by paired students t-test compared to control siRNA treated cells is indicated by * (P≤0.05) or ** (P≤0.01).
    Figure Legend Snippet: Elevated viral mRNA abundance and their enrichment in dsRNA isolated from Xrn1-depleted, VacV- infected cells Cell free lysates from NHDFs transfected with Xrn1-1 siRNA and infected with VacV (MOI=5) were prepared at 22 hpi and immunoprecipitated using J2 anti-dsRNA antibody. After treating with RNase A/T1 or RNase III, isolated RNA was analyzed by RT-qPCR using the indicated viral or cellular mRNA primers. mRNA abundances were normalized to actin and calculated relative to input (set to 1). The means of three independent experiments are plotted ± SEM. (B) Equal volumes of buffer, input lysate (IP input), or the unbound fraction (IP unbound) were dotted onto a membrane and dsRNA detected by immunoblotting to demonstrate dsRNA depletion in the unbound fraction. (C, D) NHDFs were treated with the indicated siRNAs and RNA isolated from uninfected cells (C) or 3 hpi with VACV (MOI = 5) (D). RNA was subject to RT-qPCR analysis for the indicated cellular or early viral mRNAs and each reaction product normalized to 18S rRNA and presented as the fold change relative to control siRNA-treated cells. The means of 3 independent experiments are plotted ± SEM. A significant difference by paired students t-test compared to control siRNA treated cells is indicated by * (P≤0.05) or ** (P≤0.01).

    Techniques Used: Isolation, Infection, Transfection, Immunoprecipitation, Quantitative RT-PCR

    Inhibition of protein synthesis and VacV replication by Xrn1-depletion (A) NHDFs were transfected with non-silencing (ns) control or one of two Xrn1-specific siRNAs (−1 and −2). After 3 days, total protein was collected and Xrn1 levels analyzed by immmunoblotting. Tubulin served as a loading control. (B) NHDFs treated with siRNAs as in A were infected with VacV (MOI = 5 × 10 −4 ). Infectious virus produced after 3 days was quantified by plaque assay. Means of 3 independent experiments are plotted ± SEM. ** indicates P≤0.01 by paired student’s t-test compared to control siRNA-treated samples. (C) NHDFs treated with siRNAs as in A were mock-infected or infected with VacV or UV-inactivated VacV (MOI = 5). At 18 hours post-infection (hpi), cells were metabolically pulse-labeled with [ 35 S]Met-Cys for 30 min. Total protein was collected, separated by SDS-PAGE and 35 S -labeled proteins visualized by exposing the fixed, dried gel to X-ray film. Molecular mass standards (in kDa) are shown to the right ( upper panel ). The same lysates were analyzed by immunoblotting (IB) with the indicated antibodies ( lower panel .
    Figure Legend Snippet: Inhibition of protein synthesis and VacV replication by Xrn1-depletion (A) NHDFs were transfected with non-silencing (ns) control or one of two Xrn1-specific siRNAs (−1 and −2). After 3 days, total protein was collected and Xrn1 levels analyzed by immmunoblotting. Tubulin served as a loading control. (B) NHDFs treated with siRNAs as in A were infected with VacV (MOI = 5 × 10 −4 ). Infectious virus produced after 3 days was quantified by plaque assay. Means of 3 independent experiments are plotted ± SEM. ** indicates P≤0.01 by paired student’s t-test compared to control siRNA-treated samples. (C) NHDFs treated with siRNAs as in A were mock-infected or infected with VacV or UV-inactivated VacV (MOI = 5). At 18 hours post-infection (hpi), cells were metabolically pulse-labeled with [ 35 S]Met-Cys for 30 min. Total protein was collected, separated by SDS-PAGE and 35 S -labeled proteins visualized by exposing the fixed, dried gel to X-ray film. Molecular mass standards (in kDa) are shown to the right ( upper panel ). The same lysates were analyzed by immunoblotting (IB) with the indicated antibodies ( lower panel .

    Techniques Used: Inhibition, Transfection, Infection, Produced, Plaque Assay, Metabolic Labelling, Labeling, SDS Page

    10) Product Images from "Modulation of Hepatitis C Virus RNA Abundance and Virus Release by Dispersion of Processing Bodies and Enrichment of Stress Granules"

    Article Title: Modulation of Hepatitis C Virus RNA Abundance and Virus Release by Dispersion of Processing Bodies and Enrichment of Stress Granules

    Journal: Virology

    doi: 10.1016/j.virol.2012.10.027

    Effects of depletion of P-body proteins on JFH-1 protein and RNA abundances Huh7 cells were depleted of P-body proteins and infected with JFH-1 virus. (A) Abundances of HCV NS5A and core proteins, and P-body proteins RCK/p54, Lsm1, Dcp2, Ge-1, Xrn1, Ago2, GW182, Upf1 and Exo10 during JFH-1 infection were examined by western blot analysis. Transfection reagent alone (no siRNA) and the transfection of a RISC-free siRNA were included as controls. (B) Abundances of HCV RNA and miR-122. Representative northern blots are shown. Actin mRNA and U6 snRNA were used as RNA loading controls. (C) Quantitation of HCV RNA. HCV RNA abundances were normalized to actin mRNA and to control RISC-free siRNA. Mean values and standard deviations from three independent experiments are shown.
    Figure Legend Snippet: Effects of depletion of P-body proteins on JFH-1 protein and RNA abundances Huh7 cells were depleted of P-body proteins and infected with JFH-1 virus. (A) Abundances of HCV NS5A and core proteins, and P-body proteins RCK/p54, Lsm1, Dcp2, Ge-1, Xrn1, Ago2, GW182, Upf1 and Exo10 during JFH-1 infection were examined by western blot analysis. Transfection reagent alone (no siRNA) and the transfection of a RISC-free siRNA were included as controls. (B) Abundances of HCV RNA and miR-122. Representative northern blots are shown. Actin mRNA and U6 snRNA were used as RNA loading controls. (C) Quantitation of HCV RNA. HCV RNA abundances were normalized to actin mRNA and to control RISC-free siRNA. Mean values and standard deviations from three independent experiments are shown.

    Techniques Used: Infection, Western Blot, Transfection, Northern Blot, Quantitation Assay

    11) Product Images from "Modulation of Hepatitis C Virus RNA Abundance and Virus Release by Dispersion of Processing Bodies and Enrichment of Stress Granules"

    Article Title: Modulation of Hepatitis C Virus RNA Abundance and Virus Release by Dispersion of Processing Bodies and Enrichment of Stress Granules

    Journal: Virology

    doi: 10.1016/j.virol.2012.10.027

    Effects of depletion of P-body proteins on JFH-1 protein and RNA abundances Huh7 cells were depleted of P-body proteins and infected with JFH-1 virus. (A) Abundances of HCV NS5A and core proteins, and P-body proteins RCK/p54, Lsm1, Dcp2, Ge-1, Xrn1, Ago2, GW182, Upf1 and Exo10 during JFH-1 infection were examined by western blot analysis. Transfection reagent alone (no siRNA) and the transfection of a RISC-free siRNA were included as controls. (B) Abundances of HCV RNA and miR-122. Representative northern blots are shown. Actin mRNA and U6 snRNA were used as RNA loading controls. (C) Quantitation of HCV RNA. HCV RNA abundances were normalized to actin mRNA and to control RISC-free siRNA. Mean values and standard deviations from three independent experiments are shown.
    Figure Legend Snippet: Effects of depletion of P-body proteins on JFH-1 protein and RNA abundances Huh7 cells were depleted of P-body proteins and infected with JFH-1 virus. (A) Abundances of HCV NS5A and core proteins, and P-body proteins RCK/p54, Lsm1, Dcp2, Ge-1, Xrn1, Ago2, GW182, Upf1 and Exo10 during JFH-1 infection were examined by western blot analysis. Transfection reagent alone (no siRNA) and the transfection of a RISC-free siRNA were included as controls. (B) Abundances of HCV RNA and miR-122. Representative northern blots are shown. Actin mRNA and U6 snRNA were used as RNA loading controls. (C) Quantitation of HCV RNA. HCV RNA abundances were normalized to actin mRNA and to control RISC-free siRNA. Mean values and standard deviations from three independent experiments are shown.

    Techniques Used: Infection, Western Blot, Transfection, Northern Blot, Quantitation Assay

    12) Product Images from "Human Pat1b Connects Deadenylation with mRNA Decapping and Controls the Assembly of Processing Bodies ▿Human Pat1b Connects Deadenylation with mRNA Decapping and Controls the Assembly of Processing Bodies ▿ †"

    Article Title: Human Pat1b Connects Deadenylation with mRNA Decapping and Controls the Assembly of Processing Bodies ▿Human Pat1b Connects Deadenylation with mRNA Decapping and Controls the Assembly of Processing Bodies ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00429-10

    Role of Pat1b in P-body assembly and mRNA degradation. (A) Schematic representation human Pat1b subdomains, together with interacting proteins and associated functions. All the interactions depicted are RNA independent yet may be direct or indirect. (B) Hypothetical model of the Pat1b-associated complex that connects deadenylation with mRNA decapping. The acidic region (A) associates with Rck, the AN fragment with the Ccr4-Not complex, and the N fragment with Caf1 and Dcp2-Dcp1a. The homology region (H) is required for Lsm1 binding, and the HC fragment further associates with Dcp2-Dcp1a. The HC fragment also shows weak association with Edc3, Hedls, and Xrn1.
    Figure Legend Snippet: Role of Pat1b in P-body assembly and mRNA degradation. (A) Schematic representation human Pat1b subdomains, together with interacting proteins and associated functions. All the interactions depicted are RNA independent yet may be direct or indirect. (B) Hypothetical model of the Pat1b-associated complex that connects deadenylation with mRNA decapping. The acidic region (A) associates with Rck, the AN fragment with the Ccr4-Not complex, and the N fragment with Caf1 and Dcp2-Dcp1a. The homology region (H) is required for Lsm1 binding, and the HC fragment further associates with Dcp2-Dcp1a. The HC fragment also shows weak association with Edc3, Hedls, and Xrn1.

    Techniques Used: Binding Assay

    Pat1b interacts with P-body proteins. (A) HEK293 cells were transiently transfected with vector alone, HA-tagged Pat1b, or HA-tagged Pat1a. After 1 day, cytoplasmic lysates (input) were prepared for IP with anti-HA antibody. The HA-tagged proteins as well as endogenous Rck, Hedls, Xrn1, Lsm1, Lsm4, and eIF3B were detected by Western blotting. The sizes of the molecular weight markers (in thousands) are indicated on the right. (B) HEK293 cells transiently transfected with vector alone, HA-Pat1b, or HA-Pat1b-YFP were used for IP as described in the legend for panel A. Where indicated, RNase A was added to the lysates during IP. Lanes 10 and 11 show RNA extracted from the unbound fraction and stained with ethidium bromide. (C) HA-Pat1b was immunoprecipitated with HA antibody or without antibody as a control for unspecific precipitation. (D) HA-Pat1b was immunoprecipitated and subjected to increasing NaCl concentrations prior to elution of the protein complexes. *, immunoglobulin heavy chain. (E) Endogenous Xrn1, Hedls, Rck, Lsm1, and 14-3-3 were immunoprecipitated from the cytoplasmic lysate of HEK293 cells transiently transfected with HA-Pat1b. The Western blot for Rck is not shown due to an overlapping signal from the immunoglobulin heavy chain.
    Figure Legend Snippet: Pat1b interacts with P-body proteins. (A) HEK293 cells were transiently transfected with vector alone, HA-tagged Pat1b, or HA-tagged Pat1a. After 1 day, cytoplasmic lysates (input) were prepared for IP with anti-HA antibody. The HA-tagged proteins as well as endogenous Rck, Hedls, Xrn1, Lsm1, Lsm4, and eIF3B were detected by Western blotting. The sizes of the molecular weight markers (in thousands) are indicated on the right. (B) HEK293 cells transiently transfected with vector alone, HA-Pat1b, or HA-Pat1b-YFP were used for IP as described in the legend for panel A. Where indicated, RNase A was added to the lysates during IP. Lanes 10 and 11 show RNA extracted from the unbound fraction and stained with ethidium bromide. (C) HA-Pat1b was immunoprecipitated with HA antibody or without antibody as a control for unspecific precipitation. (D) HA-Pat1b was immunoprecipitated and subjected to increasing NaCl concentrations prior to elution of the protein complexes. *, immunoglobulin heavy chain. (E) Endogenous Xrn1, Hedls, Rck, Lsm1, and 14-3-3 were immunoprecipitated from the cytoplasmic lysate of HEK293 cells transiently transfected with HA-Pat1b. The Western blot for Rck is not shown due to an overlapping signal from the immunoglobulin heavy chain.

    Techniques Used: Transfection, Plasmid Preparation, Western Blot, Molecular Weight, Staining, Immunoprecipitation

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    Bethyl rabbit anti xrn1 antibody affinity purified
    SMG6-mediated endonucleolytic cleavage is impaired when CASC3 is not present. ( A ) Schematic depiction of the globin mRNA reporter. The reporter consists of three exons (orange boxes) followed by an <t>XRN1-resistant</t> element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the second exon. ( B ) Northern blot of RNA extracted from the indicated cell lines that stably express the globin reporter. The xrFrag corresponds to the 3′ part of the reporter that is resistant to degradation by XRN1 due to the xrRNA. The cell lines in lanes 3 and 6 were additionally treated with XRN1 siRNA which results in the appearance of a 3′ degradation fragment below the full-length reporter. Reporter and 3′ fragment mRNA levels were normalized to 7SL RNA which is shown as a loading control. For the relative mRNA quantification, in each condition (WT versus CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the globin WT reporter (lanes 1 and 4). Individual data points and means are plotted from n = 3 experiments. ( C ) Schematic depiction of a TOE1 minigene reporter consisting of exons 6–8 (purple boxes) followed by a probe binding cassette (gray boxes). The reporter contains either the canonical stop codon (TOE1-WT) or PTCs in exon 7 (TOE1-PTC). ( D ) Northern blot of RNA extrac ted from the indicated cell lines treated with the indicated siRNAs stably expressing the TOE1 reporter depicted in Figure 5C . The intron between exon 6 and 7 is deleted so that the reporter is constitutively spliced to contain either a normal stop codon (TOE1-WT) or a PTC (TOE1-PTC). The mRNA levels were normalized to 7SL RNA. For the relative mRNA quantification, in each condition (WT versus CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the TOE-WT reporter (lanes 1 and 4). ( E ) Schematic depiction of the triose phosphate isomerase (TPI) mRNA reporter. The reporter consists of seven exons (blue boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the fifth exon. ( F ) Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing the either the TPI WT or TPI PTC mRNA reporter. ( G ) Quantification of the northern blot shown in Figure 5F . The reporter and 3′ fragment mRNA levels were normalized to the 7SL control. For each cell line, the mRNA levels were then normalized to the respective TPI WT reporter or 3′ fragment levels. Individual data points and means are plotted from n = 3 experiments.
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    SMG6-mediated endonucleolytic cleavage is impaired when CASC3 is not present. ( A ) Schematic depiction of the globin mRNA reporter. The reporter consists of three exons (orange boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the second exon. ( B ) Northern blot of RNA extracted from the indicated cell lines that stably express the globin reporter. The xrFrag corresponds to the 3′ part of the reporter that is resistant to degradation by XRN1 due to the xrRNA. The cell lines in lanes 3 and 6 were additionally treated with XRN1 siRNA which results in the appearance of a 3′ degradation fragment below the full-length reporter. Reporter and 3′ fragment mRNA levels were normalized to 7SL RNA which is shown as a loading control. For the relative mRNA quantification, in each condition (WT versus CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the globin WT reporter (lanes 1 and 4). Individual data points and means are plotted from n = 3 experiments. ( C ) Schematic depiction of a TOE1 minigene reporter consisting of exons 6–8 (purple boxes) followed by a probe binding cassette (gray boxes). The reporter contains either the canonical stop codon (TOE1-WT) or PTCs in exon 7 (TOE1-PTC). ( D ) Northern blot of RNA extrac ted from the indicated cell lines treated with the indicated siRNAs stably expressing the TOE1 reporter depicted in Figure 5C . The intron between exon 6 and 7 is deleted so that the reporter is constitutively spliced to contain either a normal stop codon (TOE1-WT) or a PTC (TOE1-PTC). The mRNA levels were normalized to 7SL RNA. For the relative mRNA quantification, in each condition (WT versus CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the TOE-WT reporter (lanes 1 and 4). ( E ) Schematic depiction of the triose phosphate isomerase (TPI) mRNA reporter. The reporter consists of seven exons (blue boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the fifth exon. ( F ) Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing the either the TPI WT or TPI PTC mRNA reporter. ( G ) Quantification of the northern blot shown in Figure 5F . The reporter and 3′ fragment mRNA levels were normalized to the 7SL control. For each cell line, the mRNA levels were then normalized to the respective TPI WT reporter or 3′ fragment levels. Individual data points and means are plotted from n = 3 experiments.

    Journal: Nucleic Acids Research

    Article Title: CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex

    doi: 10.1093/nar/gkaa564

    Figure Lengend Snippet: SMG6-mediated endonucleolytic cleavage is impaired when CASC3 is not present. ( A ) Schematic depiction of the globin mRNA reporter. The reporter consists of three exons (orange boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the second exon. ( B ) Northern blot of RNA extracted from the indicated cell lines that stably express the globin reporter. The xrFrag corresponds to the 3′ part of the reporter that is resistant to degradation by XRN1 due to the xrRNA. The cell lines in lanes 3 and 6 were additionally treated with XRN1 siRNA which results in the appearance of a 3′ degradation fragment below the full-length reporter. Reporter and 3′ fragment mRNA levels were normalized to 7SL RNA which is shown as a loading control. For the relative mRNA quantification, in each condition (WT versus CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the globin WT reporter (lanes 1 and 4). Individual data points and means are plotted from n = 3 experiments. ( C ) Schematic depiction of a TOE1 minigene reporter consisting of exons 6–8 (purple boxes) followed by a probe binding cassette (gray boxes). The reporter contains either the canonical stop codon (TOE1-WT) or PTCs in exon 7 (TOE1-PTC). ( D ) Northern blot of RNA extrac ted from the indicated cell lines treated with the indicated siRNAs stably expressing the TOE1 reporter depicted in Figure 5C . The intron between exon 6 and 7 is deleted so that the reporter is constitutively spliced to contain either a normal stop codon (TOE1-WT) or a PTC (TOE1-PTC). The mRNA levels were normalized to 7SL RNA. For the relative mRNA quantification, in each condition (WT versus CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the TOE-WT reporter (lanes 1 and 4). ( E ) Schematic depiction of the triose phosphate isomerase (TPI) mRNA reporter. The reporter consists of seven exons (blue boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the fifth exon. ( F ) Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing the either the TPI WT or TPI PTC mRNA reporter. ( G ) Quantification of the northern blot shown in Figure 5F . The reporter and 3′ fragment mRNA levels were normalized to the 7SL control. For each cell line, the mRNA levels were then normalized to the respective TPI WT reporter or 3′ fragment levels. Individual data points and means are plotted from n = 3 experiments.

    Article Snippet: The following antibodies were used: anti-CASC3 amino acid residues 653–703 (Bethyl Laboratories, #A302-472A-M), anti-CASC3 amino acid residues 367–470 (Atlas Antibodies, #HPA024592), anti-EIF4A3 (Genscript), anti-FLAG (Cell Signaling Technology, #14793), anti-MAGOH (Santa Cruz Biotechnology, #sc-271365), anti-PABPC1 (Cell Signaling Technology, #4992), anti-RBM8A (Atlas Antibodies, #HPA018403), anti-SMG6 (Abcam, #ab87539), anti-SMG7 (Elabscience, #E-AB-32926), anti-Tubulin (Sigma-Aldrich, #T6074), anti-UPF3B (antiserum was raised in rabbits by Eurogentech against a C-terminal fragment of UPF3B (300–483) and affinity purified), anti-V5 (QED Bioscience, #18870), anti-XRN1 (Bethyl Laboratories, #A300-443A), anti-rabbit-HRP (Jackson ImmunoResearch, #111-035-006), anti-mouse-HRP (Jackson ImmunoResearch, #115-035-003).

    Techniques: Binding Assay, Northern Blot, Stable Transfection, Expressing

    The CASC3 N-terminus promotes but is not necessary to elicit NMD. ( A ) Schematic depiction of the TPI-4MS2V5-SMG5 tethering reporter. The reporter consists of the TPI ORF (blue boxes) followed by 4 MS2 stem loops (SL). Downstream the SMG5 3′ untranslated region (UTR) is inserted to increase the size of 3′ fragments that result from cleavage at the termination codon. Reporter and 3′ fragment mRNAs can be detected via the probe binding cassette (gray boxes). ( B ) Northern blot of a tethering assay performed in HeLa Tet-Off cells. The cells stably express the tethering reporter shown in Figure 6A together with the indicated MS2V5-tagged proteins. When the cells are additionally treated with XRN1 siRNA, a 3′ degradation fragment can be detected below the full-length reporter. The reporter and 3′ fragment mRNA levels are normalized to the 7SL RNA. For the calculation of the relative mRNA levels in each condition (Luc vs. XRN1) the levels were normalized to the MS2V5-GST control (lanes 1 and 5). ( C ) Schematic depiction of CASC3 rescue protein constructs. The full-length (FL) protein consists of an N-terminal (blue), C-terminal (orange) and central SELOR domain (purple). The construct 1–480 has a C-terminal deletion, whereas in the construct 110–480 both the N- and C-terminus are truncated. Both deletion constructs were also rendered EJC-binding deficient by mutating the amino acid residues 188 and 218 (F188D, W218D). ( D ) Relative quantification of the CLN6 (top) and TOE1 (bottom) transcript isoforms by qPCR in the indicated cell lines. The V5-tagged rescue proteins expressed in the KO condition are shown schematically in Figure 6C . Rescue protein expression is confirmed in E and F. Individual data points and means are plotted from n = 3 experiments. (E and F) Western blot of samples shown in Figure 6D . The expression of rescue proteins was confirmed by an antibody against CASC3 ( E ) and an antibody recognizing the V5 tag ( F ).

    Journal: Nucleic Acids Research

    Article Title: CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex

    doi: 10.1093/nar/gkaa564

    Figure Lengend Snippet: The CASC3 N-terminus promotes but is not necessary to elicit NMD. ( A ) Schematic depiction of the TPI-4MS2V5-SMG5 tethering reporter. The reporter consists of the TPI ORF (blue boxes) followed by 4 MS2 stem loops (SL). Downstream the SMG5 3′ untranslated region (UTR) is inserted to increase the size of 3′ fragments that result from cleavage at the termination codon. Reporter and 3′ fragment mRNAs can be detected via the probe binding cassette (gray boxes). ( B ) Northern blot of a tethering assay performed in HeLa Tet-Off cells. The cells stably express the tethering reporter shown in Figure 6A together with the indicated MS2V5-tagged proteins. When the cells are additionally treated with XRN1 siRNA, a 3′ degradation fragment can be detected below the full-length reporter. The reporter and 3′ fragment mRNA levels are normalized to the 7SL RNA. For the calculation of the relative mRNA levels in each condition (Luc vs. XRN1) the levels were normalized to the MS2V5-GST control (lanes 1 and 5). ( C ) Schematic depiction of CASC3 rescue protein constructs. The full-length (FL) protein consists of an N-terminal (blue), C-terminal (orange) and central SELOR domain (purple). The construct 1–480 has a C-terminal deletion, whereas in the construct 110–480 both the N- and C-terminus are truncated. Both deletion constructs were also rendered EJC-binding deficient by mutating the amino acid residues 188 and 218 (F188D, W218D). ( D ) Relative quantification of the CLN6 (top) and TOE1 (bottom) transcript isoforms by qPCR in the indicated cell lines. The V5-tagged rescue proteins expressed in the KO condition are shown schematically in Figure 6C . Rescue protein expression is confirmed in E and F. Individual data points and means are plotted from n = 3 experiments. (E and F) Western blot of samples shown in Figure 6D . The expression of rescue proteins was confirmed by an antibody against CASC3 ( E ) and an antibody recognizing the V5 tag ( F ).

    Article Snippet: The following antibodies were used: anti-CASC3 amino acid residues 653–703 (Bethyl Laboratories, #A302-472A-M), anti-CASC3 amino acid residues 367–470 (Atlas Antibodies, #HPA024592), anti-EIF4A3 (Genscript), anti-FLAG (Cell Signaling Technology, #14793), anti-MAGOH (Santa Cruz Biotechnology, #sc-271365), anti-PABPC1 (Cell Signaling Technology, #4992), anti-RBM8A (Atlas Antibodies, #HPA018403), anti-SMG6 (Abcam, #ab87539), anti-SMG7 (Elabscience, #E-AB-32926), anti-Tubulin (Sigma-Aldrich, #T6074), anti-UPF3B (antiserum was raised in rabbits by Eurogentech against a C-terminal fragment of UPF3B (300–483) and affinity purified), anti-V5 (QED Bioscience, #18870), anti-XRN1 (Bethyl Laboratories, #A300-443A), anti-rabbit-HRP (Jackson ImmunoResearch, #111-035-006), anti-mouse-HRP (Jackson ImmunoResearch, #115-035-003).

    Techniques: Binding Assay, Northern Blot, Stable Transfection, Construct, Real-time Polymerase Chain Reaction, Expressing, Western Blot

    XRN1 also regulates ncRNAs in SAOS-2 cells. A) Assessment of transcript proportions affected by XRN1 depletion relative to the genome wide proportions detected in our sequencing data. HOTAIR highlighted in black, grey=no change, red=post-transcriptionally upregulated, orange=transcriptionally upregulated, purple=post-transcriptionally downregulated, blue=transcriptionally downregulated, green=no intron data. B) Scatter plot of changes in expression of all ncRNAs detected in our sequencing data. C-E) Boxplots of C) expression D) GC content and E) length (bp) of ncRNAs in our data. Grouped by their nature of change in expression and compared to the genome average as detected in our data set.

    Journal: bioRxiv

    Article Title: Genome-wide analyses of XRN1-sensitive targets in osteosarcoma cells identifies disease-relevant transcripts containing G-rich motifs

    doi: 10.1101/2020.08.11.246249

    Figure Lengend Snippet: XRN1 also regulates ncRNAs in SAOS-2 cells. A) Assessment of transcript proportions affected by XRN1 depletion relative to the genome wide proportions detected in our sequencing data. HOTAIR highlighted in black, grey=no change, red=post-transcriptionally upregulated, orange=transcriptionally upregulated, purple=post-transcriptionally downregulated, blue=transcriptionally downregulated, green=no intron data. B) Scatter plot of changes in expression of all ncRNAs detected in our sequencing data. C-E) Boxplots of C) expression D) GC content and E) length (bp) of ncRNAs in our data. Grouped by their nature of change in expression and compared to the genome average as detected in our data set.

    Article Snippet: Primary antibodies used were Mouse anti-GAPDH (1:10,000, Abcam #ab8245), Mouse anti-Tubulin (1:2000, Sigma #T9026) and Rabbit anti-XRN1 (1:2000, Bethyl Labs #A300-443A).

    Techniques: Genome Wide, Sequencing, Expressing

    Direct XRN1 targets possess G-rich motifs. A/B) MEME analysis of 3’UTR of 103 mRNAs reveal A) GU rich 18 sites across 10 unique transcripts and B) G-rich 233 sites across 69 unique transcripts, motifs which may confer XRN1 sensitivity. C) Similar analysis of 30 ncRNAs post-transcriptionally upregulated in XRN1-depleted SAOS-2 cells reveals a similar G-rich motif to that observed in B (89 sites across 21 unique transcripts).

    Journal: bioRxiv

    Article Title: Genome-wide analyses of XRN1-sensitive targets in osteosarcoma cells identifies disease-relevant transcripts containing G-rich motifs

    doi: 10.1101/2020.08.11.246249

    Figure Lengend Snippet: Direct XRN1 targets possess G-rich motifs. A/B) MEME analysis of 3’UTR of 103 mRNAs reveal A) GU rich 18 sites across 10 unique transcripts and B) G-rich 233 sites across 69 unique transcripts, motifs which may confer XRN1 sensitivity. C) Similar analysis of 30 ncRNAs post-transcriptionally upregulated in XRN1-depleted SAOS-2 cells reveals a similar G-rich motif to that observed in B (89 sites across 21 unique transcripts).

    Article Snippet: Primary antibodies used were Mouse anti-GAPDH (1:10,000, Abcam #ab8245), Mouse anti-Tubulin (1:2000, Sigma #T9026) and Rabbit anti-XRN1 (1:2000, Bethyl Labs #A300-443A).

    Techniques:

    XRN1 is downregulated in osteo- and Ewing sarcoma. A) qRT-PCR quantification of XRN1 mRNA expression across osteosarcoma (OS) cell lines in comparison to the HOb control cell line, normalised to HPRT1 . Error bars represent SEM, n≥5. B) qRT-PCR quantification of pre-XRN1 across osteosarcoma cell lines in comparison to the HOb control cell line, normalised to HPRT1 . Error bars represent SEM, n≥6, C) qRT-PCR quantification of XRN1 across osteosarcoma patient samples in comparison to the HOb control cell line, normalised to PES1 . Error bars represent SEM, n≥5, p=0.0296. Red = samples from hip and femur, blue=samples from scapula or humerus and green=unknown origin. D) Representative Western blot and graphical analysis showing expression of XRN1 protein in osteosarcoma cells as a proportion of that expressed in HOb control cells. Error bars represent SEM, n≥4. E) qRT-PCR quantification of XRN1 mRNA expression in Ewing sarcoma (EWS) cell lines in comparison to the HOb control cell line, normalised to GAPDH . Error bars represent SEM, n≥6. F) Representative Western blot and graphical analysis showing expression of XRN1 protein in Ewing sarcoma cells as a proportion of that expressed in Hob control cells Error bars represent SEM, n≥4. For all figures ****=p

    Journal: bioRxiv

    Article Title: Genome-wide analyses of XRN1-sensitive targets in osteosarcoma cells identifies disease-relevant transcripts containing G-rich motifs

    doi: 10.1101/2020.08.11.246249

    Figure Lengend Snippet: XRN1 is downregulated in osteo- and Ewing sarcoma. A) qRT-PCR quantification of XRN1 mRNA expression across osteosarcoma (OS) cell lines in comparison to the HOb control cell line, normalised to HPRT1 . Error bars represent SEM, n≥5. B) qRT-PCR quantification of pre-XRN1 across osteosarcoma cell lines in comparison to the HOb control cell line, normalised to HPRT1 . Error bars represent SEM, n≥6, C) qRT-PCR quantification of XRN1 across osteosarcoma patient samples in comparison to the HOb control cell line, normalised to PES1 . Error bars represent SEM, n≥5, p=0.0296. Red = samples from hip and femur, blue=samples from scapula or humerus and green=unknown origin. D) Representative Western blot and graphical analysis showing expression of XRN1 protein in osteosarcoma cells as a proportion of that expressed in HOb control cells. Error bars represent SEM, n≥4. E) qRT-PCR quantification of XRN1 mRNA expression in Ewing sarcoma (EWS) cell lines in comparison to the HOb control cell line, normalised to GAPDH . Error bars represent SEM, n≥6. F) Representative Western blot and graphical analysis showing expression of XRN1 protein in Ewing sarcoma cells as a proportion of that expressed in Hob control cells Error bars represent SEM, n≥4. For all figures ****=p

    Article Snippet: Primary antibodies used were Mouse anti-GAPDH (1:10,000, Abcam #ab8245), Mouse anti-Tubulin (1:2000, Sigma #T9026) and Rabbit anti-XRN1 (1:2000, Bethyl Labs #A300-443A).

    Techniques: Quantitative RT-PCR, Expressing, Western Blot

    Gene Ontology analysis of differentially expressed transcripts. A) Gene ontology analysis using DAVID and Biological processes level “BPFAT” at highest stringency on all differentially expressed transcripts in XRN1-depleted SAOS-2 cells. B) As A, but enriched biological processes assessed in individual groups of misregulated transcripts.

    Journal: bioRxiv

    Article Title: Genome-wide analyses of XRN1-sensitive targets in osteosarcoma cells identifies disease-relevant transcripts containing G-rich motifs

    doi: 10.1101/2020.08.11.246249

    Figure Lengend Snippet: Gene Ontology analysis of differentially expressed transcripts. A) Gene ontology analysis using DAVID and Biological processes level “BPFAT” at highest stringency on all differentially expressed transcripts in XRN1-depleted SAOS-2 cells. B) As A, but enriched biological processes assessed in individual groups of misregulated transcripts.

    Article Snippet: Primary antibodies used were Mouse anti-GAPDH (1:10,000, Abcam #ab8245), Mouse anti-Tubulin (1:2000, Sigma #T9026) and Rabbit anti-XRN1 (1:2000, Bethyl Labs #A300-443A).

    Techniques:

    XRN1-sensitive transcripts show specific transcript characteristics. Boxplots of A) expression, B) GC content, C) translational efficiency, D) 5’UTR length, E) coding sequence (CDS) length and F) 3’UTR length of all transcripts within our data set. Grouped by their nature of change in expression and compared to the genome average as detected in our data set. Translational efficiency calculated as ribosome protected footprint FPKM/total RNA FPKM for each transcript.

    Journal: bioRxiv

    Article Title: Genome-wide analyses of XRN1-sensitive targets in osteosarcoma cells identifies disease-relevant transcripts containing G-rich motifs

    doi: 10.1101/2020.08.11.246249

    Figure Lengend Snippet: XRN1-sensitive transcripts show specific transcript characteristics. Boxplots of A) expression, B) GC content, C) translational efficiency, D) 5’UTR length, E) coding sequence (CDS) length and F) 3’UTR length of all transcripts within our data set. Grouped by their nature of change in expression and compared to the genome average as detected in our data set. Translational efficiency calculated as ribosome protected footprint FPKM/total RNA FPKM for each transcript.

    Article Snippet: Primary antibodies used were Mouse anti-GAPDH (1:10,000, Abcam #ab8245), Mouse anti-Tubulin (1:2000, Sigma #T9026) and Rabbit anti-XRN1 (1:2000, Bethyl Labs #A300-443A).

    Techniques: Expressing, Sequencing

    Overview of RNA-sequencing of XRN1-depleted SAOS-2 cells. A) Up- (red) and downregulated (blue) transcripts based on initial edgeR differential expression using genes as a counting method in featureCounts. B) Scrambled vs XRN1 knockdown FPKM based demonstrating differentially expressed transcripts using both gene and exon counting. Exon FPKM used for direct comparison with intron counting. Grey=no change, red=post-transcriptionally upregulated, orange=transcriptionally upregulated, purple=post-transcriptionally downregulated, blue=transcriptionally downregulated, green=no intron data. C) Differentially expressed transcripts when counting intron mapping reads allowing differentiation between transcriptional and post-transcriptional changes represented in B. Legend as in B. D) MA plot representing fold change in XRN1-depeleted SAOS-2 cells vs transcript expression in control cells coloured by nature of change. Legend as in B. E) Volcano plot demonstrating statistical information of all expressed transcripts. Legend as in B.

    Journal: bioRxiv

    Article Title: Genome-wide analyses of XRN1-sensitive targets in osteosarcoma cells identifies disease-relevant transcripts containing G-rich motifs

    doi: 10.1101/2020.08.11.246249

    Figure Lengend Snippet: Overview of RNA-sequencing of XRN1-depleted SAOS-2 cells. A) Up- (red) and downregulated (blue) transcripts based on initial edgeR differential expression using genes as a counting method in featureCounts. B) Scrambled vs XRN1 knockdown FPKM based demonstrating differentially expressed transcripts using both gene and exon counting. Exon FPKM used for direct comparison with intron counting. Grey=no change, red=post-transcriptionally upregulated, orange=transcriptionally upregulated, purple=post-transcriptionally downregulated, blue=transcriptionally downregulated, green=no intron data. C) Differentially expressed transcripts when counting intron mapping reads allowing differentiation between transcriptional and post-transcriptional changes represented in B. Legend as in B. D) MA plot representing fold change in XRN1-depeleted SAOS-2 cells vs transcript expression in control cells coloured by nature of change. Legend as in B. E) Volcano plot demonstrating statistical information of all expressed transcripts. Legend as in B.

    Article Snippet: Primary antibodies used were Mouse anti-GAPDH (1:10,000, Abcam #ab8245), Mouse anti-Tubulin (1:2000, Sigma #T9026) and Rabbit anti-XRN1 (1:2000, Bethyl Labs #A300-443A).

    Techniques: RNA Sequencing Assay, Expressing

    XRN1 knockdown in SAOS-2 cells does not result in observable phenotypes. A) Successful knockdown of XRN1 in SAOS-2 cells using RNAi 24 hours post transfection. Scr samples treated with 20pmol scrambled siRNA and KD cells treated with 20pmol XRN1 siRNA. Error bars represent SEM, ***=p=0.0008. B) Quantification and representative images (40x objective) of the BrdU proliferation assay. Error bars represent SEM, n≥25, p=0.7938, scale bar=50µM. C) WST-1 assay at 24hr time intervals following transfection with either Scrambled (Scr) or XRN1 (KD) siRNA. Error bar represent SEM, n=3. D) Caspase Glo 3/4 assay at 24hr time intervals following transfection with either Scrambled (Scr) or XRN1 (KD) siRNA. Error bar represent SEM, n=3. E) Quantification and representative images (20x objective) of transwell migration assay 6hrs, 24hrs or 30hrs post seeding. Seeding was performed 24hrs post transfection with either Scrambled (Scr) or XRN1 (KD) siRNA. Error bars represent SEM, n=4, p > 0.05, scale bar=100µM. F) Knockdown of XRN1 does not affect nascent translation rates. Quantification of Puromycin incorporation or XRN1 expression (normalised to GAPDH relative to its own scrambled partner) 24hrs post transfection in cells treated with either Scrambled (Scr) or XRN1 (KD) siRNA. Error bars represent SEM, ***=p=0.0003, ns=p=0.7432, n=5.

    Journal: bioRxiv

    Article Title: Genome-wide analyses of XRN1-sensitive targets in osteosarcoma cells identifies disease-relevant transcripts containing G-rich motifs

    doi: 10.1101/2020.08.11.246249

    Figure Lengend Snippet: XRN1 knockdown in SAOS-2 cells does not result in observable phenotypes. A) Successful knockdown of XRN1 in SAOS-2 cells using RNAi 24 hours post transfection. Scr samples treated with 20pmol scrambled siRNA and KD cells treated with 20pmol XRN1 siRNA. Error bars represent SEM, ***=p=0.0008. B) Quantification and representative images (40x objective) of the BrdU proliferation assay. Error bars represent SEM, n≥25, p=0.7938, scale bar=50µM. C) WST-1 assay at 24hr time intervals following transfection with either Scrambled (Scr) or XRN1 (KD) siRNA. Error bar represent SEM, n=3. D) Caspase Glo 3/4 assay at 24hr time intervals following transfection with either Scrambled (Scr) or XRN1 (KD) siRNA. Error bar represent SEM, n=3. E) Quantification and representative images (20x objective) of transwell migration assay 6hrs, 24hrs or 30hrs post seeding. Seeding was performed 24hrs post transfection with either Scrambled (Scr) or XRN1 (KD) siRNA. Error bars represent SEM, n=4, p > 0.05, scale bar=100µM. F) Knockdown of XRN1 does not affect nascent translation rates. Quantification of Puromycin incorporation or XRN1 expression (normalised to GAPDH relative to its own scrambled partner) 24hrs post transfection in cells treated with either Scrambled (Scr) or XRN1 (KD) siRNA. Error bars represent SEM, ***=p=0.0003, ns=p=0.7432, n=5.

    Article Snippet: Primary antibodies used were Mouse anti-GAPDH (1:10,000, Abcam #ab8245), Mouse anti-Tubulin (1:2000, Sigma #T9026) and Rabbit anti-XRN1 (1:2000, Bethyl Labs #A300-443A).

    Techniques: Transfection, Proliferation Assay, WST-1 Assay, Transwell Migration Assay, Expressing

    Dcp2 targeting of RNAs correlates with P-body enrichment and is enhanced by m 6 A modification. (a-e) Boxplots quantifying differential expression of RNAs after DDX6 silencing in HEK293T cells relative to control (data from Courel et al. , 2019) (a), translation efficiency (data from Sidrauski et al. , 2015) (b), 5′ UTR length (c), 3′ UTR length (reference datasets from Khong et al. , 2017) (d), and RNA enrichment after XRN1 knockout in HEK293T cells (data from Chang et al ., 2019) (e) for each of the three classes of mRNA stability changes in DCP2 KO versus WT HEK293T cells. Statistical significance is calculated using Mann-Whitney U test. Ns, not significant (p > 0.05); ***P

    Journal: Biochemistry

    Article Title: Global profiling of cellular substrates of human Dcp2

    doi: 10.1021/acs.biochem.0c00069

    Figure Lengend Snippet: Dcp2 targeting of RNAs correlates with P-body enrichment and is enhanced by m 6 A modification. (a-e) Boxplots quantifying differential expression of RNAs after DDX6 silencing in HEK293T cells relative to control (data from Courel et al. , 2019) (a), translation efficiency (data from Sidrauski et al. , 2015) (b), 5′ UTR length (c), 3′ UTR length (reference datasets from Khong et al. , 2017) (d), and RNA enrichment after XRN1 knockout in HEK293T cells (data from Chang et al ., 2019) (e) for each of the three classes of mRNA stability changes in DCP2 KO versus WT HEK293T cells. Statistical significance is calculated using Mann-Whitney U test. Ns, not significant (p > 0.05); ***P

    Article Snippet: Primary antibodies used for Western blotting and/or immunofluorescence were as follows: rabbit polyclonal anti-Dcp2 (Novus Biologicals, NBP1-41070); mouse monoclonal anti-beta actin (Invitrogen, BA3R); rabbit polyclonal anti-Xrn1 (Bethyl, A300-443A; Sigma, PLA0105); rabbit monoclonal anti-Msi2 (Abcam, ab76148); rabbit monoclonal anti-Dcp1a Alexa Fluor 647 (Abcam, ab209946).

    Techniques: Modification, Expressing, Knock-Out, MANN-WHITNEY

    Validation of transcript stabilization in DCP2 KO cells. (a) The stabilities of selected Dcp2 targets were measured using qRT-PCR in DCP2 KO versus WT HEK293T cells treated with actinomycin D for the indicated times. Half-lives (95% CI) were obtained from linear regression analysis, and significance was determined using two-tailed t -test of the slope of regression lines. Error bars represent mean ± s.d. n =4 biological replicates for HOXA13 , GATA6 and CCNT1 and n in WT, XRN1 KO and XRN1/DCP2 double knockout (DKO) HEK293T cell lines was performed to assay decapping of selected Dcp2 targets. Significance was analyzed by ANOVA linear regression (total RNA) or at individual time points (splint ligation). Error bars represent mean ± s.d. n =4 biological replicates. P-values are denoted by asterisks; *P

    Journal: Biochemistry

    Article Title: Global profiling of cellular substrates of human Dcp2

    doi: 10.1021/acs.biochem.0c00069

    Figure Lengend Snippet: Validation of transcript stabilization in DCP2 KO cells. (a) The stabilities of selected Dcp2 targets were measured using qRT-PCR in DCP2 KO versus WT HEK293T cells treated with actinomycin D for the indicated times. Half-lives (95% CI) were obtained from linear regression analysis, and significance was determined using two-tailed t -test of the slope of regression lines. Error bars represent mean ± s.d. n =4 biological replicates for HOXA13 , GATA6 and CCNT1 and n in WT, XRN1 KO and XRN1/DCP2 double knockout (DKO) HEK293T cell lines was performed to assay decapping of selected Dcp2 targets. Significance was analyzed by ANOVA linear regression (total RNA) or at individual time points (splint ligation). Error bars represent mean ± s.d. n =4 biological replicates. P-values are denoted by asterisks; *P

    Article Snippet: Primary antibodies used for Western blotting and/or immunofluorescence were as follows: rabbit polyclonal anti-Dcp2 (Novus Biologicals, NBP1-41070); mouse monoclonal anti-beta actin (Invitrogen, BA3R); rabbit polyclonal anti-Xrn1 (Bethyl, A300-443A; Sigma, PLA0105); rabbit monoclonal anti-Msi2 (Abcam, ab76148); rabbit monoclonal anti-Dcp1a Alexa Fluor 647 (Abcam, ab209946).

    Techniques: Quantitative RT-PCR, Two Tailed Test, Double Knockout, Ligation

    SMG6-mediated endocleavage is impaired when CASC3 is not present. A : Schematic depiction of the globin mRNA reporter. The reporter consists of three exons (orange boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the second exon. B : Northern blot of RNA extracted from the indicated cell lines that stably express the globin reporter. The xrFrag corresponds to the 3′ part of the reporter that is resistant to degradation by XRN1 due to the xrRNA. The cell lines in lane 3 and 6 were additionally treated with XRN1 siRNA which results in the appearance of a 3′ degradation fragment below the full-length reporter. Reporter and 3′ fragment mRNA levels were normalized to 7SL RNA which is shown as a loading control. For the relative mRNA quantification, in each condition (WT vs. CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the globin WT reporter (lanes 1 and 4). Individual data points and means are plotted from n=3 experiments. C : Schematic depiction of the TOE1 minigene reporter consisting of exons 6-8 (purple boxes) followed by a probe binding cassette (gray boxes). The reporter can be spliced to either contain the canonical stop codon (bottom right) or, by usage of an alternative 3′ splice site, a PTC in exon 7 (top right). D : Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing the TOE1 minigene reporter. The 3′ fragment levels were first normalized to the 7SL RNA loading control and for every cell line the XRN1 knockdown condition to the condition without XRN1 knockdown (n=2). E : Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing a prespliced variant of the TOE1 minigene reporter depicted in Figure 5C. The intron between exon 6 and 7 is deleted so that the reporter is constitutively spliced to contain either a normal stop codon (TOE1-WT) or a PTC (TOE1-PTC). The mRNA levels were normalized to 7SL RNA. For the relative mRNA quantification, in each condition (WT vs. CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the TOE-WT reporter (lanes 1 and 4). F : Schematic depiction of the triose phosphate isomerase (TPI) mRNA reporter. The reporter consists of seven exons (blue boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the fifth exon. G : Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing the either the TPI WT or TPI PTC mRNA reporter. The reporter and 3′ fragment mRNA levels were normalized to the 7SL control. For each cell line, the mRNA levels were then normalized to the respective TPI WT reporter or 3′ fragment levels. Individual data points and means are plotted from n=3 experiments.

    Journal: bioRxiv

    Article Title: CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex

    doi: 10.1101/811018

    Figure Lengend Snippet: SMG6-mediated endocleavage is impaired when CASC3 is not present. A : Schematic depiction of the globin mRNA reporter. The reporter consists of three exons (orange boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the second exon. B : Northern blot of RNA extracted from the indicated cell lines that stably express the globin reporter. The xrFrag corresponds to the 3′ part of the reporter that is resistant to degradation by XRN1 due to the xrRNA. The cell lines in lane 3 and 6 were additionally treated with XRN1 siRNA which results in the appearance of a 3′ degradation fragment below the full-length reporter. Reporter and 3′ fragment mRNA levels were normalized to 7SL RNA which is shown as a loading control. For the relative mRNA quantification, in each condition (WT vs. CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the globin WT reporter (lanes 1 and 4). Individual data points and means are plotted from n=3 experiments. C : Schematic depiction of the TOE1 minigene reporter consisting of exons 6-8 (purple boxes) followed by a probe binding cassette (gray boxes). The reporter can be spliced to either contain the canonical stop codon (bottom right) or, by usage of an alternative 3′ splice site, a PTC in exon 7 (top right). D : Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing the TOE1 minigene reporter. The 3′ fragment levels were first normalized to the 7SL RNA loading control and for every cell line the XRN1 knockdown condition to the condition without XRN1 knockdown (n=2). E : Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing a prespliced variant of the TOE1 minigene reporter depicted in Figure 5C. The intron between exon 6 and 7 is deleted so that the reporter is constitutively spliced to contain either a normal stop codon (TOE1-WT) or a PTC (TOE1-PTC). The mRNA levels were normalized to 7SL RNA. For the relative mRNA quantification, in each condition (WT vs. CASC3 KO with KD) the reporter and 3′ fragment levels were normalized to the TOE-WT reporter (lanes 1 and 4). F : Schematic depiction of the triose phosphate isomerase (TPI) mRNA reporter. The reporter consists of seven exons (blue boxes) followed by an XRN1-resistant element (xrRNA) and a probe binding cassette (gray boxes). The PTC reporter contains a premature termination codon (PTC) in the fifth exon. G : Northern blot of RNA extracted from the indicated cell lines treated with the indicated siRNAs stably expressing the either the TPI WT or TPI PTC mRNA reporter. The reporter and 3′ fragment mRNA levels were normalized to the 7SL control. For each cell line, the mRNA levels were then normalized to the respective TPI WT reporter or 3′ fragment levels. Individual data points and means are plotted from n=3 experiments.

    Article Snippet: The following antibodies were used: anti-CASC3 amino acid residues 653-703 (Bethyl Laboratories, #A302-472A-M), anti-CASC3 amino acid residues 367-470 (Atlas Antibodies, #HPA024592), anti-EIF4A3 (Genscript), anti-FLAG (Cell Signaling Technology, #14793), anti-RBM8A (Atlas Antibodies, #HPA018403), anti-SMG6 (Abcam, #ab87539), anti-SMG7 (Elabscience, #E-AB-32926), anti-Tubulin (Sigma-Aldrich, #T6074), anti-V5 (QED Bioscience, #18870), anti-XRN1 (Bethyl Laboratories, #A300-443A), anti-rabbit-HRP (Jackson ImmunoResearch, #111-035-006), anti-mouse-HRP (Jackson ImmunoResearch, #115-035-003).

    Techniques: Binding Assay, Northern Blot, Stable Transfection, Expressing, Variant Assay

    The CASC3 N-terminus promotes but is not necessary to elicit NMD. A : Schematic depiction of the TPI-MS2V5-SMG5 tethering reporter. The reporter consists of the TPI ORF (blue boxes) followed by 4 MS2 stem loops (SL). Downstream the SMG5 3′ untranslated region (UTR) is inserted to increase the size of 3′ fragments that result from cleavage at the termination codon. Reporter and 3′ fragment mRNAs can be detected via the probe binding cassette (gray boxes). B : Northern blot of a tethering assay performed in HeLa Tet-Off cells. The cells stably express the tethering reporter shown in Figure 6A together with the indicated MS2V5-tagged proteins. When the cells are additionally treated with XRN1 siRNA, a 3′ degradation fragment can be detected below the full-length reporter. The reporter and 3′ fragment mRNA levels are normalized to the 7SL RNA. For the calculation of the relative mRNA levels in each condition (Luc vs. XRN1) the levels were normalized to the MS2V5-GST control (lanes 1 and 5). C : Schematic depiction of CASC3 rescue protein constructs. The full-length (FL) protein consists of an N-terminal (blue), C-terminal (orange) and central SELOR domain (purple). The construct 1-480 has a C-terminal deletion, whereas in the construct 110-480 both the N- and C-terminus are truncated. Both deletion constructs were also rendered EJC-binding deficient by mutating the amino acid residues 188 and 218 (F188D, W218D). D : Relative quantification of the CLN6 (top) and TOE1 (bottom) transcript isoforms by qPCR in the indicated cell lines. The V5-tagged rescue proteins expressed in the KO condition are shown schematically in Figure 6C. Rescue protein expression is confirmed in Figure 6E and F. Individual data points and means are plotted from n=3 experiments. E and F: Western blot of samples shown in Figure 6D. The expression of rescue proteins was confirmed by an antibody against CASC3 (E) and an antibody recognizing the V5 tag (F).

    Journal: bioRxiv

    Article Title: CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex

    doi: 10.1101/811018

    Figure Lengend Snippet: The CASC3 N-terminus promotes but is not necessary to elicit NMD. A : Schematic depiction of the TPI-MS2V5-SMG5 tethering reporter. The reporter consists of the TPI ORF (blue boxes) followed by 4 MS2 stem loops (SL). Downstream the SMG5 3′ untranslated region (UTR) is inserted to increase the size of 3′ fragments that result from cleavage at the termination codon. Reporter and 3′ fragment mRNAs can be detected via the probe binding cassette (gray boxes). B : Northern blot of a tethering assay performed in HeLa Tet-Off cells. The cells stably express the tethering reporter shown in Figure 6A together with the indicated MS2V5-tagged proteins. When the cells are additionally treated with XRN1 siRNA, a 3′ degradation fragment can be detected below the full-length reporter. The reporter and 3′ fragment mRNA levels are normalized to the 7SL RNA. For the calculation of the relative mRNA levels in each condition (Luc vs. XRN1) the levels were normalized to the MS2V5-GST control (lanes 1 and 5). C : Schematic depiction of CASC3 rescue protein constructs. The full-length (FL) protein consists of an N-terminal (blue), C-terminal (orange) and central SELOR domain (purple). The construct 1-480 has a C-terminal deletion, whereas in the construct 110-480 both the N- and C-terminus are truncated. Both deletion constructs were also rendered EJC-binding deficient by mutating the amino acid residues 188 and 218 (F188D, W218D). D : Relative quantification of the CLN6 (top) and TOE1 (bottom) transcript isoforms by qPCR in the indicated cell lines. The V5-tagged rescue proteins expressed in the KO condition are shown schematically in Figure 6C. Rescue protein expression is confirmed in Figure 6E and F. Individual data points and means are plotted from n=3 experiments. E and F: Western blot of samples shown in Figure 6D. The expression of rescue proteins was confirmed by an antibody against CASC3 (E) and an antibody recognizing the V5 tag (F).

    Article Snippet: The following antibodies were used: anti-CASC3 amino acid residues 653-703 (Bethyl Laboratories, #A302-472A-M), anti-CASC3 amino acid residues 367-470 (Atlas Antibodies, #HPA024592), anti-EIF4A3 (Genscript), anti-FLAG (Cell Signaling Technology, #14793), anti-RBM8A (Atlas Antibodies, #HPA018403), anti-SMG6 (Abcam, #ab87539), anti-SMG7 (Elabscience, #E-AB-32926), anti-Tubulin (Sigma-Aldrich, #T6074), anti-V5 (QED Bioscience, #18870), anti-XRN1 (Bethyl Laboratories, #A300-443A), anti-rabbit-HRP (Jackson ImmunoResearch, #111-035-006), anti-mouse-HRP (Jackson ImmunoResearch, #115-035-003).

    Techniques: Binding Assay, Northern Blot, Stable Transfection, Construct, Real-time Polymerase Chain Reaction, Expressing, Western Blot