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  • 99
    Thermo Fisher rnase
    CPSF6 affects MVC alternative RNA processing. (A) Transcription profile of MVC showing the P6 promoter, transcription starting site (TSS), splice donors (D) and acceptors (A), and proximal [(pA)p], and distal [(pA)d] polyadenylation sites. The annotated nucleotides delineate the boundaries of the transcription landmarks indicated within the MVC genome. The position of the <t>RNase</t> protection probes, 2A/3D (nt 2344 to 2550), 3A (nt 2910 to 3110), and (pA)p (nt 3107 to 3333), are indicated. NP1 and NS-66 mRNA species polyadenylated at (pA)p or (pA)d are shown. (B) RPAs of total RNA extracted 48 h following transfection of 293FT or CPSF6(-)293T cells with MVC Rep-Cap (lanes 1 and 2), IMVC-WT (lanes 3 and 4), and IMVC-5X (lanes 5 and 6) using the (pA)p probe. The sizes of the probe and various protected bands are shown on the left. The protected bands representing RNA species extending through (pA)p to (pA)d or cleaved at the various cleavage sites as described for (pA)d and (pA)p are indicated to the right. Quantifications below show the ratio of (pA)p/(pA)d RNAs from transfected CPSF6 knockout cells compared to the level in 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (C) Immunoprecipitation analysis reveals the association of CPSF6 and MVC NP1 in 293FT cells. Equal amounts of cell lysates were <t>immunoprecipitated</t> (IP) using protein-G magnetic beads (lane 2) or anti-HA magnetic beads (lane 3), as described in Materials and Methods, followed by immunoblotting with antibodies against CPSF6 and HA-tagged NP1. (D) RPAs of total RNA as described for panel B using the 3A probe. The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species (3Aunspl, unspliced at the third intron acceptor [3A]; 3Aspl, third intron acceptor spliced) are indicated to the right. Quantifications below show the ratio of spliced/unspliced RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (E) RPAs of total RNA as described for panels B and D using the 2A/3D probe (nt 2910 to 3100). The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species [(pA)d, read-through of the second intron acceptor (2A) and third intron donor (3D); 2Aspl/3Dun, the second intron acceptor spliced but third intron unspliced; 2Aspl/3Dspl, both second intron acceptor and third intron spliced] are indicated to the right. Quantifications below show the ratio of 2Aspl/3Dspl to read-through RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (F) 293FT or CPSF6(-)293T cells transfected with constructs described for panels B, D, and E were harvested and analyzed by immunoblotting using antibodies directed against CPSF6, NS, and NP1 (the individual epitopes are described in Materials and Methods). Immunoblotting for β-actin was used as loading control.
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    Thermo Fisher rnase out ribonuclease inhibitor
    CPSF6 affects MVC alternative RNA processing. (A) Transcription profile of MVC showing the P6 promoter, transcription starting site (TSS), splice donors (D) and acceptors (A), and proximal [(pA)p], and distal [(pA)d] polyadenylation sites. The annotated nucleotides delineate the boundaries of the transcription landmarks indicated within the MVC genome. The position of the <t>RNase</t> protection probes, 2A/3D (nt 2344 to 2550), 3A (nt 2910 to 3110), and (pA)p (nt 3107 to 3333), are indicated. NP1 and NS-66 mRNA species polyadenylated at (pA)p or (pA)d are shown. (B) RPAs of total RNA extracted 48 h following transfection of 293FT or CPSF6(-)293T cells with MVC Rep-Cap (lanes 1 and 2), IMVC-WT (lanes 3 and 4), and IMVC-5X (lanes 5 and 6) using the (pA)p probe. The sizes of the probe and various protected bands are shown on the left. The protected bands representing RNA species extending through (pA)p to (pA)d or cleaved at the various cleavage sites as described for (pA)d and (pA)p are indicated to the right. Quantifications below show the ratio of (pA)p/(pA)d RNAs from transfected CPSF6 knockout cells compared to the level in 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (C) Immunoprecipitation analysis reveals the association of CPSF6 and MVC NP1 in 293FT cells. Equal amounts of cell lysates were <t>immunoprecipitated</t> (IP) using protein-G magnetic beads (lane 2) or anti-HA magnetic beads (lane 3), as described in Materials and Methods, followed by immunoblotting with antibodies against CPSF6 and HA-tagged NP1. (D) RPAs of total RNA as described for panel B using the 3A probe. The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species (3Aunspl, unspliced at the third intron acceptor [3A]; 3Aspl, third intron acceptor spliced) are indicated to the right. Quantifications below show the ratio of spliced/unspliced RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (E) RPAs of total RNA as described for panels B and D using the 2A/3D probe (nt 2910 to 3100). The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species [(pA)d, read-through of the second intron acceptor (2A) and third intron donor (3D); 2Aspl/3Dun, the second intron acceptor spliced but third intron unspliced; 2Aspl/3Dspl, both second intron acceptor and third intron spliced] are indicated to the right. Quantifications below show the ratio of 2Aspl/3Dspl to read-through RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (F) 293FT or CPSF6(-)293T cells transfected with constructs described for panels B, D, and E were harvested and analyzed by immunoblotting using antibodies directed against CPSF6, NS, and NP1 (the individual epitopes are described in Materials and Methods). Immunoblotting for β-actin was used as loading control.
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    CPSF6 affects MVC alternative RNA processing. (A) Transcription profile of MVC showing the P6 promoter, transcription starting site (TSS), splice donors (D) and acceptors (A), and proximal [(pA)p], and distal [(pA)d] polyadenylation sites. The annotated nucleotides delineate the boundaries of the transcription landmarks indicated within the MVC genome. The position of the <t>RNase</t> protection probes, 2A/3D (nt 2344 to 2550), 3A (nt 2910 to 3110), and (pA)p (nt 3107 to 3333), are indicated. NP1 and NS-66 mRNA species polyadenylated at (pA)p or (pA)d are shown. (B) RPAs of total RNA extracted 48 h following transfection of 293FT or CPSF6(-)293T cells with MVC Rep-Cap (lanes 1 and 2), IMVC-WT (lanes 3 and 4), and IMVC-5X (lanes 5 and 6) using the (pA)p probe. The sizes of the probe and various protected bands are shown on the left. The protected bands representing RNA species extending through (pA)p to (pA)d or cleaved at the various cleavage sites as described for (pA)d and (pA)p are indicated to the right. Quantifications below show the ratio of (pA)p/(pA)d RNAs from transfected CPSF6 knockout cells compared to the level in 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (C) Immunoprecipitation analysis reveals the association of CPSF6 and MVC NP1 in 293FT cells. Equal amounts of cell lysates were <t>immunoprecipitated</t> (IP) using protein-G magnetic beads (lane 2) or anti-HA magnetic beads (lane 3), as described in Materials and Methods, followed by immunoblotting with antibodies against CPSF6 and HA-tagged NP1. (D) RPAs of total RNA as described for panel B using the 3A probe. The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species (3Aunspl, unspliced at the third intron acceptor [3A]; 3Aspl, third intron acceptor spliced) are indicated to the right. Quantifications below show the ratio of spliced/unspliced RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (E) RPAs of total RNA as described for panels B and D using the 2A/3D probe (nt 2910 to 3100). The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species [(pA)d, read-through of the second intron acceptor (2A) and third intron donor (3D); 2Aspl/3Dun, the second intron acceptor spliced but third intron unspliced; 2Aspl/3Dspl, both second intron acceptor and third intron spliced] are indicated to the right. Quantifications below show the ratio of 2Aspl/3Dspl to read-through RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (F) 293FT or CPSF6(-)293T cells transfected with constructs described for panels B, D, and E were harvested and analyzed by immunoblotting using antibodies directed against CPSF6, NS, and NP1 (the individual epitopes are described in Materials and Methods). Immunoblotting for β-actin was used as loading control.
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    CPSF6 affects MVC alternative RNA processing. (A) Transcription profile of MVC showing the P6 promoter, transcription starting site (TSS), splice donors (D) and acceptors (A), and proximal [(pA)p], and distal [(pA)d] polyadenylation sites. The annotated nucleotides delineate the boundaries of the transcription landmarks indicated within the MVC genome. The position of the <t>RNase</t> protection probes, 2A/3D (nt 2344 to 2550), 3A (nt 2910 to 3110), and (pA)p (nt 3107 to 3333), are indicated. NP1 and NS-66 mRNA species polyadenylated at (pA)p or (pA)d are shown. (B) RPAs of total RNA extracted 48 h following transfection of 293FT or CPSF6(-)293T cells with MVC Rep-Cap (lanes 1 and 2), IMVC-WT (lanes 3 and 4), and IMVC-5X (lanes 5 and 6) using the (pA)p probe. The sizes of the probe and various protected bands are shown on the left. The protected bands representing RNA species extending through (pA)p to (pA)d or cleaved at the various cleavage sites as described for (pA)d and (pA)p are indicated to the right. Quantifications below show the ratio of (pA)p/(pA)d RNAs from transfected CPSF6 knockout cells compared to the level in 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (C) Immunoprecipitation analysis reveals the association of CPSF6 and MVC NP1 in 293FT cells. Equal amounts of cell lysates were <t>immunoprecipitated</t> (IP) using protein-G magnetic beads (lane 2) or anti-HA magnetic beads (lane 3), as described in Materials and Methods, followed by immunoblotting with antibodies against CPSF6 and HA-tagged NP1. (D) RPAs of total RNA as described for panel B using the 3A probe. The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species (3Aunspl, unspliced at the third intron acceptor [3A]; 3Aspl, third intron acceptor spliced) are indicated to the right. Quantifications below show the ratio of spliced/unspliced RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (E) RPAs of total RNA as described for panels B and D using the 2A/3D probe (nt 2910 to 3100). The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species [(pA)d, read-through of the second intron acceptor (2A) and third intron donor (3D); 2Aspl/3Dun, the second intron acceptor spliced but third intron unspliced; 2Aspl/3Dspl, both second intron acceptor and third intron spliced] are indicated to the right. Quantifications below show the ratio of 2Aspl/3Dspl to read-through RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (F) 293FT or CPSF6(-)293T cells transfected with constructs described for panels B, D, and E were harvested and analyzed by immunoblotting using antibodies directed against CPSF6, NS, and NP1 (the individual epitopes are described in Materials and Methods). Immunoblotting for β-actin was used as loading control.
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    Ligand-induced conformational changes. In-line probing of a 5′-AlexaFluor 647-labeled Bs PreQ 1 -RS RNA and b 5′-AlexaFluor 647-labeled Tt PreQ 1 -RS RNA after treatment with 1 at increasing concentrations or a DMSO control in the absence (–) or presence (+) of 1 m m MgCl 2 . Treatment with PreQ 1 at a concentration of 10 μ m is used as a positive control. OH and T1 are a partial alkaline hydrolysis ladder and <t>ribonuclease</t> T1 digestion, respectively. Arrows designate nucleotide positions where the cleavage efficiency was significantly altered by compound treatment (blue) or preQ 1 treatment (red)
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    Ligand-induced conformational changes. In-line probing of a 5′-AlexaFluor 647-labeled Bs PreQ 1 -RS RNA and b 5′-AlexaFluor 647-labeled Tt PreQ 1 -RS RNA after treatment with 1 at increasing concentrations or a DMSO control in the absence (–) or presence (+) of 1 m m MgCl 2 . Treatment with PreQ 1 at a concentration of 10 μ m is used as a positive control. OH and T1 are a partial alkaline hydrolysis ladder and <t>ribonuclease</t> T1 digestion, respectively. Arrows designate nucleotide positions where the cleavage efficiency was significantly altered by compound treatment (blue) or preQ 1 treatment (red)
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    Thermo Fisher rpa ii rnase protection kit
    Ligand-induced conformational changes. In-line probing of a 5′-AlexaFluor 647-labeled Bs PreQ 1 -RS RNA and b 5′-AlexaFluor 647-labeled Tt PreQ 1 -RS RNA after treatment with 1 at increasing concentrations or a DMSO control in the absence (–) or presence (+) of 1 m m MgCl 2 . Treatment with PreQ 1 at a concentration of 10 μ m is used as a positive control. OH and T1 are a partial alkaline hydrolysis ladder and <t>ribonuclease</t> T1 digestion, respectively. Arrows designate nucleotide positions where the cleavage efficiency was significantly altered by compound treatment (blue) or preQ 1 treatment (red)
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    Ligand-induced conformational changes. In-line probing of a 5′-AlexaFluor 647-labeled Bs PreQ 1 -RS RNA and b 5′-AlexaFluor 647-labeled Tt PreQ 1 -RS RNA after treatment with 1 at increasing concentrations or a DMSO control in the absence (–) or presence (+) of 1 m m MgCl 2 . Treatment with PreQ 1 at a concentration of 10 μ m is used as a positive control. OH and T1 are a partial alkaline hydrolysis ladder and <t>ribonuclease</t> T1 digestion, respectively. Arrows designate nucleotide positions where the cleavage efficiency was significantly altered by compound treatment (blue) or preQ 1 treatment (red)
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    Ligand-induced conformational changes. In-line probing of a 5′-AlexaFluor 647-labeled Bs PreQ 1 -RS RNA and b 5′-AlexaFluor 647-labeled Tt PreQ 1 -RS RNA after treatment with 1 at increasing concentrations or a DMSO control in the absence (–) or presence (+) of 1 m m MgCl 2 . Treatment with PreQ 1 at a concentration of 10 μ m is used as a positive control. OH and T1 are a partial alkaline hydrolysis ladder and <t>ribonuclease</t> T1 digestion, respectively. Arrows designate nucleotide positions where the cleavage efficiency was significantly altered by compound treatment (blue) or preQ 1 treatment (red)
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    Ligand-induced conformational changes. In-line probing of a 5′-AlexaFluor 647-labeled Bs PreQ 1 -RS RNA and b 5′-AlexaFluor 647-labeled Tt PreQ 1 -RS RNA after treatment with 1 at increasing concentrations or a DMSO control in the absence (–) or presence (+) of 1 m m MgCl 2 . Treatment with PreQ 1 at a concentration of 10 μ m is used as a positive control. OH and T1 are a partial alkaline hydrolysis ladder and <t>ribonuclease</t> T1 digestion, respectively. Arrows designate nucleotide positions where the cleavage efficiency was significantly altered by compound treatment (blue) or preQ 1 treatment (red)
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    Image Search Results


    CPSF6 affects MVC alternative RNA processing. (A) Transcription profile of MVC showing the P6 promoter, transcription starting site (TSS), splice donors (D) and acceptors (A), and proximal [(pA)p], and distal [(pA)d] polyadenylation sites. The annotated nucleotides delineate the boundaries of the transcription landmarks indicated within the MVC genome. The position of the RNase protection probes, 2A/3D (nt 2344 to 2550), 3A (nt 2910 to 3110), and (pA)p (nt 3107 to 3333), are indicated. NP1 and NS-66 mRNA species polyadenylated at (pA)p or (pA)d are shown. (B) RPAs of total RNA extracted 48 h following transfection of 293FT or CPSF6(-)293T cells with MVC Rep-Cap (lanes 1 and 2), IMVC-WT (lanes 3 and 4), and IMVC-5X (lanes 5 and 6) using the (pA)p probe. The sizes of the probe and various protected bands are shown on the left. The protected bands representing RNA species extending through (pA)p to (pA)d or cleaved at the various cleavage sites as described for (pA)d and (pA)p are indicated to the right. Quantifications below show the ratio of (pA)p/(pA)d RNAs from transfected CPSF6 knockout cells compared to the level in 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (C) Immunoprecipitation analysis reveals the association of CPSF6 and MVC NP1 in 293FT cells. Equal amounts of cell lysates were immunoprecipitated (IP) using protein-G magnetic beads (lane 2) or anti-HA magnetic beads (lane 3), as described in Materials and Methods, followed by immunoblotting with antibodies against CPSF6 and HA-tagged NP1. (D) RPAs of total RNA as described for panel B using the 3A probe. The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species (3Aunspl, unspliced at the third intron acceptor [3A]; 3Aspl, third intron acceptor spliced) are indicated to the right. Quantifications below show the ratio of spliced/unspliced RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (E) RPAs of total RNA as described for panels B and D using the 2A/3D probe (nt 2910 to 3100). The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species [(pA)d, read-through of the second intron acceptor (2A) and third intron donor (3D); 2Aspl/3Dun, the second intron acceptor spliced but third intron unspliced; 2Aspl/3Dspl, both second intron acceptor and third intron spliced] are indicated to the right. Quantifications below show the ratio of 2Aspl/3Dspl to read-through RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (F) 293FT or CPSF6(-)293T cells transfected with constructs described for panels B, D, and E were harvested and analyzed by immunoblotting using antibodies directed against CPSF6, NS, and NP1 (the individual epitopes are described in Materials and Methods). Immunoblotting for β-actin was used as loading control.

    Journal: Journal of Virology

    Article Title: Minute Virus of Canines NP1 Protein Interacts with the Cellular Factor CPSF6 To Regulate Viral Alternative RNA Processing

    doi: 10.1128/JVI.01530-18

    Figure Lengend Snippet: CPSF6 affects MVC alternative RNA processing. (A) Transcription profile of MVC showing the P6 promoter, transcription starting site (TSS), splice donors (D) and acceptors (A), and proximal [(pA)p], and distal [(pA)d] polyadenylation sites. The annotated nucleotides delineate the boundaries of the transcription landmarks indicated within the MVC genome. The position of the RNase protection probes, 2A/3D (nt 2344 to 2550), 3A (nt 2910 to 3110), and (pA)p (nt 3107 to 3333), are indicated. NP1 and NS-66 mRNA species polyadenylated at (pA)p or (pA)d are shown. (B) RPAs of total RNA extracted 48 h following transfection of 293FT or CPSF6(-)293T cells with MVC Rep-Cap (lanes 1 and 2), IMVC-WT (lanes 3 and 4), and IMVC-5X (lanes 5 and 6) using the (pA)p probe. The sizes of the probe and various protected bands are shown on the left. The protected bands representing RNA species extending through (pA)p to (pA)d or cleaved at the various cleavage sites as described for (pA)d and (pA)p are indicated to the right. Quantifications below show the ratio of (pA)p/(pA)d RNAs from transfected CPSF6 knockout cells compared to the level in 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (C) Immunoprecipitation analysis reveals the association of CPSF6 and MVC NP1 in 293FT cells. Equal amounts of cell lysates were immunoprecipitated (IP) using protein-G magnetic beads (lane 2) or anti-HA magnetic beads (lane 3), as described in Materials and Methods, followed by immunoblotting with antibodies against CPSF6 and HA-tagged NP1. (D) RPAs of total RNA as described for panel B using the 3A probe. The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species (3Aunspl, unspliced at the third intron acceptor [3A]; 3Aspl, third intron acceptor spliced) are indicated to the right. Quantifications below show the ratio of spliced/unspliced RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (E) RPAs of total RNA as described for panels B and D using the 2A/3D probe (nt 2910 to 3100). The sizes of the probe and various protected bands are shown on the left. Bands representing RNA species [(pA)d, read-through of the second intron acceptor (2A) and third intron donor (3D); 2Aspl/3Dun, the second intron acceptor spliced but third intron unspliced; 2Aspl/3Dspl, both second intron acceptor and third intron spliced] are indicated to the right. Quantifications below show the ratio of 2Aspl/3Dspl to read-through RNAs from transfected CPSF6(-)293T cells compared to the value for 293FT cells, which was set to 1. Standard errors were derived from at least three independent experiments. (F) 293FT or CPSF6(-)293T cells transfected with constructs described for panels B, D, and E were harvested and analyzed by immunoblotting using antibodies directed against CPSF6, NS, and NP1 (the individual epitopes are described in Materials and Methods). Immunoblotting for β-actin was used as loading control.

    Article Snippet: After centrifugation at 14,000 rpm for 30 min at 4°C, the supernatant was divided into two samples of equal volumes, treated with RNase, and immunoprecipitated with either protein G magnetic beads (Pierce/ThermoFisher) or anti-HA magnetic beads (Pierce/ThermoFisher) overnight at 4°C.

    Techniques: Transfection, Knock-Out, Derivative Assay, Immunoprecipitation, Magnetic Beads, Construct

    Complementary-template reverse-transcription (CT-RT) of single-stranded DNA primers reverse-transcribed from FFPE-RNA. ( a ) RNA extracted from FFPE tissue is reverse-transcribed, the mRNA/DNA duplex is filtered on an YM-50 column and the DNA is single-stranded with RNase-H and column purified. The 5′-NB-Oligo-dA (24) -cT7-3′ (complementary to the T7 promoter) is annealed to the FFPE-cDNA primers. ( b ) Total RNA from universal human reference (UHR, Stratagene) is amplified using the Sense-Amp cRNA amplification kit from Genisphere to provide RNA with the same orientation as messenger RNA ( 32 ). ( c ) Single-stranded DNA primers are hybridized to their sense-RNA template between 70 and 42°C for 90 min. The hybridized products are reverse-transcribed by a process described as CT-RT. The restored FFPE-cDNAs are doubled stranded and transcribed in vitro using T7 polymerase. (See Supplementary Data for technical description of points 1 through 6.)

    Journal: Nucleic Acids Research

    Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

    doi: 10.1093/nar/gkm510

    Figure Lengend Snippet: Complementary-template reverse-transcription (CT-RT) of single-stranded DNA primers reverse-transcribed from FFPE-RNA. ( a ) RNA extracted from FFPE tissue is reverse-transcribed, the mRNA/DNA duplex is filtered on an YM-50 column and the DNA is single-stranded with RNase-H and column purified. The 5′-NB-Oligo-dA (24) -cT7-3′ (complementary to the T7 promoter) is annealed to the FFPE-cDNA primers. ( b ) Total RNA from universal human reference (UHR, Stratagene) is amplified using the Sense-Amp cRNA amplification kit from Genisphere to provide RNA with the same orientation as messenger RNA ( 32 ). ( c ) Single-stranded DNA primers are hybridized to their sense-RNA template between 70 and 42°C for 90 min. The hybridized products are reverse-transcribed by a process described as CT-RT. The restored FFPE-cDNAs are doubled stranded and transcribed in vitro using T7 polymerase. (See Supplementary Data for technical description of points 1 through 6.)

    Article Snippet: The aRNA was incubated in the presence of 2.67 μl of random primers (3 μg/μl) from Invitrogen in a final volume of 19 μl at 70°C for 10 min, spun down and put on ice for 5 min. Labeling reactions were performed by adding 8 μl of 5× first strand buffer, 4 μl of 0.1 M DTT, 4 μl of dNTP labeling mix (2.5 mM of each), 4 μl of 25 nM Cy3-labeled deoxyuridine triphosphate (Cy3-dUTP) for cRNA amplified from UHR (reference) or 4 μl of 25 nM Cy5-labeled deoxyuridine triphosphate (Cy5-dUTP; Amersham Pharmacia Biotech, NJ, USA) for cRNA from our samples, 1 μl of RNase-out at 40 U/μl (Invitrogen), 1.5 μl of Superscript II reverse-transcriptase 200 U/μl (Invitrogen) and incubated at 42°C for 1 h. After 1 h of incubation, 1.5 μl of Superscript II reverse-transcriptase was added for another 60 min at 42°C.

    Techniques: Formalin-fixed Paraffin-Embedded, Purification, Amplification, In Vitro

    RNase H knockout increases the expansion rate of transcribed (GAA) n repeats. ( A ) Schematic of the GAA;CD system to select for repeat expansion events in yeast. A total of 100 (GAA) n repeats were cloned into an artificially split URA3 gene such that expansion events abrogate splicing and result in resistance to 5-FOA. ARS306 : autonomously replicating sequence on Chr III. P GAL1 : Galactose inducible promoter. TRP1 : auxotrophic marker for selection of strains bearing the construct. ‘Up’ indicates the region amplified by primers used for RT-qPCR. ( B ) (GAA) 100 expansion rates in strains bearing the construct in A. Error bars represent 95% confidence intervals. Cells were grown non-selectively on glucose (low) or galactose (high) to modulate transcription. ‘**’ Indicates non-overlapping 95% confidence intervals compared to WT under the same conditions. Numbers indicate fold change in expansion rate in rnh1Δ, rnh201Δ strains compared to WT.

    Journal: Nucleic Acids Research

    Article Title: RNA–DNA hybrids promote the expansion of Friedreich's ataxia (GAA)n repeats via break-induced replication

    doi: 10.1093/nar/gky099

    Figure Lengend Snippet: RNase H knockout increases the expansion rate of transcribed (GAA) n repeats. ( A ) Schematic of the GAA;CD system to select for repeat expansion events in yeast. A total of 100 (GAA) n repeats were cloned into an artificially split URA3 gene such that expansion events abrogate splicing and result in resistance to 5-FOA. ARS306 : autonomously replicating sequence on Chr III. P GAL1 : Galactose inducible promoter. TRP1 : auxotrophic marker for selection of strains bearing the construct. ‘Up’ indicates the region amplified by primers used for RT-qPCR. ( B ) (GAA) 100 expansion rates in strains bearing the construct in A. Error bars represent 95% confidence intervals. Cells were grown non-selectively on glucose (low) or galactose (high) to modulate transcription. ‘**’ Indicates non-overlapping 95% confidence intervals compared to WT under the same conditions. Numbers indicate fold change in expansion rate in rnh1Δ, rnh201Δ strains compared to WT.

    Article Snippet: Following the RT reaction, samples were treated with 0.5U RNase H at 37°C for 20 min. Quantitative PCR (qPCR) was performed in 10 μl volumes using SYBR Select Master Mix (ThermoFisher) on a QuantStudio 6 Flex RTPCR system (Applied Biosystems) in 384-well plates with four technical replicates per sample.

    Techniques: Knock-Out, Clone Assay, Sequencing, Marker, Selection, Construct, Amplification, Quantitative RT-PCR

    Altering the direction of transcription-replication collisions or inverting the repetitive run alone does not change the effect of RNase H deletion on repeat expansion rate. ( A ) Schematic of our GAA;HO construct where transcription and replication are oriented head-on. (TTC) 100 serves as the lagging strand template and (GAA) 100 remains on the transcriptional coding strand. ( B ) Repeat expansion rates in strains bearing different constructs for selection of repeat expansion events. Error bars represent 95% confidence intervals. Cells were grown non-selectively on glucose (low) or galactose (high) to modulate transcription. Numbers indicate fold increase in expansion rate due to galactose induction. ( C ) Schematic of our TTC;CD construct, in which only the repeats have been flipped such that (TTC) 100 now serves as both the lagging strand template and the transcriptional coding strand. ( D ) Repeat expansion rates in WT and RNase H deficient strains bearing our different constructs for selecting for repeat expansion events. Error bars represent 95% confidence intervals. Cells were grown non-selectively on glucose (low) or galactose (high) to modulate transcription. ‘**’ Indicates non-overlapping 95% confidence intervals compared to corresponding WT strain and conditions ‘*’ Indicates non-overlapping 84% confidence intervals compared to corresponding WT strain and conditions. Numbers indicate fold change in expansion rate in rnh1Δ, rnh201Δ strains compared to WT.

    Journal: Nucleic Acids Research

    Article Title: RNA–DNA hybrids promote the expansion of Friedreich's ataxia (GAA)n repeats via break-induced replication

    doi: 10.1093/nar/gky099

    Figure Lengend Snippet: Altering the direction of transcription-replication collisions or inverting the repetitive run alone does not change the effect of RNase H deletion on repeat expansion rate. ( A ) Schematic of our GAA;HO construct where transcription and replication are oriented head-on. (TTC) 100 serves as the lagging strand template and (GAA) 100 remains on the transcriptional coding strand. ( B ) Repeat expansion rates in strains bearing different constructs for selection of repeat expansion events. Error bars represent 95% confidence intervals. Cells were grown non-selectively on glucose (low) or galactose (high) to modulate transcription. Numbers indicate fold increase in expansion rate due to galactose induction. ( C ) Schematic of our TTC;CD construct, in which only the repeats have been flipped such that (TTC) 100 now serves as both the lagging strand template and the transcriptional coding strand. ( D ) Repeat expansion rates in WT and RNase H deficient strains bearing our different constructs for selecting for repeat expansion events. Error bars represent 95% confidence intervals. Cells were grown non-selectively on glucose (low) or galactose (high) to modulate transcription. ‘**’ Indicates non-overlapping 95% confidence intervals compared to corresponding WT strain and conditions ‘*’ Indicates non-overlapping 84% confidence intervals compared to corresponding WT strain and conditions. Numbers indicate fold change in expansion rate in rnh1Δ, rnh201Δ strains compared to WT.

    Article Snippet: Following the RT reaction, samples were treated with 0.5U RNase H at 37°C for 20 min. Quantitative PCR (qPCR) was performed in 10 μl volumes using SYBR Select Master Mix (ThermoFisher) on a QuantStudio 6 Flex RTPCR system (Applied Biosystems) in 384-well plates with four technical replicates per sample.

    Techniques: Construct, Selection

    RNase H knockout does not affect the expansion rate of non-transcribed (GAA) n repeats. ( A ) Schematic of our system to select for expansion of non-transcribed repeats in yeast. A total of 100 (GAA) n repeats were cloned into the region between the UAS and TSS of a GAL1 promoter driving expression of the CAN1 gene. Repeat expansion events diminish promoter activity allowing for selection on media containing canvanine. ARS306 : autonomously replicating sequence on Chr III. TRP1 : auxotrophic marker for selection of strains bearing the construct. ( B ) (GAA) 100 expansion rates in strains bearing the construct in A. Error bars represent 95% confidence intervals. Cells were grown non-selectively on glucose (low) or galactose (high) to modulate transcription. Differences between WT and rnh1Δ, rnh201Δ strains were not significant.

    Journal: Nucleic Acids Research

    Article Title: RNA–DNA hybrids promote the expansion of Friedreich's ataxia (GAA)n repeats via break-induced replication

    doi: 10.1093/nar/gky099

    Figure Lengend Snippet: RNase H knockout does not affect the expansion rate of non-transcribed (GAA) n repeats. ( A ) Schematic of our system to select for expansion of non-transcribed repeats in yeast. A total of 100 (GAA) n repeats were cloned into the region between the UAS and TSS of a GAL1 promoter driving expression of the CAN1 gene. Repeat expansion events diminish promoter activity allowing for selection on media containing canvanine. ARS306 : autonomously replicating sequence on Chr III. TRP1 : auxotrophic marker for selection of strains bearing the construct. ( B ) (GAA) 100 expansion rates in strains bearing the construct in A. Error bars represent 95% confidence intervals. Cells were grown non-selectively on glucose (low) or galactose (high) to modulate transcription. Differences between WT and rnh1Δ, rnh201Δ strains were not significant.

    Article Snippet: Following the RT reaction, samples were treated with 0.5U RNase H at 37°C for 20 min. Quantitative PCR (qPCR) was performed in 10 μl volumes using SYBR Select Master Mix (ThermoFisher) on a QuantStudio 6 Flex RTPCR system (Applied Biosystems) in 384-well plates with four technical replicates per sample.

    Techniques: Knock-Out, Clone Assay, Expressing, Activity Assay, Selection, Sequencing, Marker, Construct

    RNase H knockout increases (GAA) n contraction rate via mechanisms that differ from expansion. ( A ) Schematic of our construct for selecting for contraction events of transcribed (GAA) 128 repeats. Our URA3 construct from Figure 1A was altered such that strains start out Ura − and become Ura + after repeat contraction. ( B ) Schematic of our construct for selecting for contraction events on the non-transcribed (GAA) 128 repeat. The negative selective CAN1 gene from the cassette in Figure 2A was replaced by the positive selection HIS3 marker. Strains with the (GAA) 128 repeat are His − . Contraction of the repeats between the UAS and TSS leads to promoter activation and a His + phenotype. ( C ) (GAA) 128 contraction rates in strains bearing the construct in A. Error bars represent 95% confidence intervals. ‘**’ Indicates non-overlapping 95% confidence intervals compared to WT. ‘∧∧’ Indicates non-overlapping 95% confidence intervals compared to WT grown on glucose. ( D ) (GAA) 128 contraction rates in strains bearing the construct in B. Error bars represent 95% confidence intervals. ‘∧∧’ Indicates non-overlapping 95% confidence intervals compared to WT grown on glucose.

    Journal: Nucleic Acids Research

    Article Title: RNA–DNA hybrids promote the expansion of Friedreich's ataxia (GAA)n repeats via break-induced replication

    doi: 10.1093/nar/gky099

    Figure Lengend Snippet: RNase H knockout increases (GAA) n contraction rate via mechanisms that differ from expansion. ( A ) Schematic of our construct for selecting for contraction events of transcribed (GAA) 128 repeats. Our URA3 construct from Figure 1A was altered such that strains start out Ura − and become Ura + after repeat contraction. ( B ) Schematic of our construct for selecting for contraction events on the non-transcribed (GAA) 128 repeat. The negative selective CAN1 gene from the cassette in Figure 2A was replaced by the positive selection HIS3 marker. Strains with the (GAA) 128 repeat are His − . Contraction of the repeats between the UAS and TSS leads to promoter activation and a His + phenotype. ( C ) (GAA) 128 contraction rates in strains bearing the construct in A. Error bars represent 95% confidence intervals. ‘**’ Indicates non-overlapping 95% confidence intervals compared to WT. ‘∧∧’ Indicates non-overlapping 95% confidence intervals compared to WT grown on glucose. ( D ) (GAA) 128 contraction rates in strains bearing the construct in B. Error bars represent 95% confidence intervals. ‘∧∧’ Indicates non-overlapping 95% confidence intervals compared to WT grown on glucose.

    Article Snippet: Following the RT reaction, samples were treated with 0.5U RNase H at 37°C for 20 min. Quantitative PCR (qPCR) was performed in 10 μl volumes using SYBR Select Master Mix (ThermoFisher) on a QuantStudio 6 Flex RTPCR system (Applied Biosystems) in 384-well plates with four technical replicates per sample.

    Techniques: Knock-Out, Construct, Selection, Marker, Activation Assay

    Ligand-induced conformational changes. In-line probing of a 5′-AlexaFluor 647-labeled Bs PreQ 1 -RS RNA and b 5′-AlexaFluor 647-labeled Tt PreQ 1 -RS RNA after treatment with 1 at increasing concentrations or a DMSO control in the absence (–) or presence (+) of 1 m m MgCl 2 . Treatment with PreQ 1 at a concentration of 10 μ m is used as a positive control. OH and T1 are a partial alkaline hydrolysis ladder and ribonuclease T1 digestion, respectively. Arrows designate nucleotide positions where the cleavage efficiency was significantly altered by compound treatment (blue) or preQ 1 treatment (red)

    Journal: Nature Communications

    Article Title: Synthetic ligands for PreQ1 riboswitches provide structural and mechanistic insights into targeting RNA tertiary structure

    doi: 10.1038/s41467-019-09493-3

    Figure Lengend Snippet: Ligand-induced conformational changes. In-line probing of a 5′-AlexaFluor 647-labeled Bs PreQ 1 -RS RNA and b 5′-AlexaFluor 647-labeled Tt PreQ 1 -RS RNA after treatment with 1 at increasing concentrations or a DMSO control in the absence (–) or presence (+) of 1 m m MgCl 2 . Treatment with PreQ 1 at a concentration of 10 μ m is used as a positive control. OH and T1 are a partial alkaline hydrolysis ladder and ribonuclease T1 digestion, respectively. Arrows designate nucleotide positions where the cleavage efficiency was significantly altered by compound treatment (blue) or preQ 1 treatment (red)

    Article Snippet: Alkaline hydrolysis was performed in 50 mm NaHCO3 , pH 9.0 at 95 °C for 5 min. Ribonuclease T1 digestion was carried out with 0.1 U of ribonuclease T1 (Ambion) in 20 mm Tris, pH 7.5, 50 mm NaCl, 0.1 mm MgCl2 at room temperature for 20 min.

    Techniques: Labeling, Concentration Assay, Positive Control