rabbit anti son antibody  (Novus Biologicals)

Journal: bioRxiv

Article Title: Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

doi: 10.1101/2024.02.29.581881

Figure Lengend Snippet: (A) Scheme of ARTR-seq. Specific speckle scaffold protein is immunostained by primary and secondary antibodies sequentially. pAG-RTase is then allowed to bind to the antibody to initiate reverse transcription in situ. The generated biotinylated cDNAs are collected and prepared for sequencing. (B) Representative image showing colocalization of pAG-RTase labeled with Alexa Fluor 647 (AF647) (red), secondary antibody labeled with Alexa Fluor 568 (AF568, yellow) against anti-SON primary antibody, and generated biotinylated-cDNA detected by Alexa Fluor 488 (AF488) labeled antibody against biotin (green). Scale bar: 3 µm. (C) Venn diagram of overlapped speckle-enriched genes identified through targeting SON and SRRM2 in HeLa cells using Method 1 or Method 2. Speckle-enriched transcripts are defined by I NSE >2 and adjusted p-value < 0.05. (D) Genome tracks showing ARTR-seq reads generated from targeting SON or SRRM2 proteins, and from control samples without primary antibody, mapped to gene locus encoding lncRNA MALAT1 . (E) RNA FISH images showing speckle-enriched LAMA5 transcript in comparison with speckle non-enriched P4HB transcript. RNA FISH probes are labeled with AF647 (red). Nucleus was stained with DAPI. Scale bar: 10 μm. (F) Correlation between speckle partition coefficient (R NS/NU ) measured by RNA FISH imaging and I NSE values determined by ARTR-seq using Method 1.

Article Snippet: Cells were immunostained with rabbit anti-SON antibody (1:200 dilution, Novus) or mouse anti-SRRM2 antibody (1:200 dilution, Sigma Aldrich) for 1h at room temperature followed by 3 times wash with DPBS.

Techniques: Reverse Transcription, In Situ, Generated, Sequencing, Labeling, Control, Comparison, Staining, Imaging

Journal: bioRxiv

Article Title: Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

doi: 10.1101/2024.02.29.581881

Figure Lengend Snippet: (A) Fraction of unspliced introns (EI/(EI+EE)) calculated from the number of reads mapping to exon-intron boundary (EI) and the number of reads mapping to exon-exon junction (EE) in polyA RNA-seq, nuclear RNA-seq, ARTR-seq without antibody and ARTR-seq with SON antibody. (B) Alternative estimation of the fraction of unspliced introns (IN/(IN+EE)) calculated using reads mapping to intronic positions within 100 nucleotides of splice sites (IN) instead of EI. (C) Ratio of IN reads to EI reads in polyA RNA-seq, nuclear RNA-seq, ARTR-seq without antibody and ARTR-seq with SON antibody. The two replicates of RNA-seq were calculated individually. Each bar with error bars in (A)-(C) reports mean and standard deviation of the two replicates. (D) Violin plot showing the speckle size immunostained with SRRM2 antibody under NT Plad B treatment. “N” indicates the total number of speckles in each data set. (E-F) 2D histogram showing the correlation of I NSE(intron) (E) or I NSE(exon) (F) between NT and Plad B treatment conditions in HeLa cells. Genes were categorized into three Groups (Group A, B, C) and depicted in (F). (G-H) Violin plot comparing I NSE(exon) (G) or I NSE(intron) (H) values among Group A, B, and C genes under NT and Plad B treatment conditions. P-values calculated with unpaired t-test are reported above each violin plot. “N” reports the total number of genes in each comparison. (I) GO analysis for speckle-enriched Group A and Group B genes, and non-speckle-enriched Group C gene. The analysis is performed using g:Profiler , using the background consisting of all Group A, B and C genes. GO terms in biological processes (BP) and cellular compartment (CC) were identified.

Article Snippet: Cells were immunostained with rabbit anti-SON antibody (1:200 dilution, Novus) or mouse anti-SRRM2 antibody (1:200 dilution, Sigma Aldrich) for 1h at room temperature followed by 3 times wash with DPBS.

Techniques: RNA Sequencing Assay, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

doi: 10.1101/2024.02.29.581881

Figure Lengend Snippet: (A) Scatter plot showing randomly selected genes from Group A, B and C genes, and corresponding I NSE(exon) under NT and Plad B treatment conditions in HeLa cells. (B) Genome tracks showing polyA RNA-seq (pink) and nuclear RNA-seq (blue) under NT and Plad B treatment conditions for selected genes: THOC6 in Group A gene, TUBB4B in Group B gene, and NCL in Group C gene. Selected efficiently spliced or inefficiently spliced introns for RT-PCR assay are highlighted in cyan and red boxes respectively. Genome tracks of other selected genes for RT-PCR assays are shown in Figure S8. (C) Schematic description of the RT-PCR assay. After reverse transcription of extracted total RNA, primers located on two adjacent exons of selected introns were used for amplification and the PCR products were analyzed by electrophoresis. (D) Representative immunofluorescence images showing nuclear speckles upon SON / SRRM2 double knockdown (KD) and treated with control siRNA (siC). Nuclear speckles were stained with AF488 labeled antibody against SRRM2 antibody (blue); and nuclei were stained with DAPI (grey). Scale bar: 10 μm. (E) Histogram of SRRM2 immunofluorescence intensity distribution of cells with double knockdown (KD) or treatment with control siRNA (siC). (F) Violin plot showing the number of speckles per cell for KD and siC treatment. Total number of cells included in each data set is indicated by “N” in (E) and (F). P-values calculated with unpaired t-tests are reported above each violin plot. (G) Representative electrophoresis analysis of RT-PCR products from THOC6 , TUBB4B and NCL upon KD and siC treatment. Gels of other selected genes for RT-PCR assays are shown in Figure S8. Gels were imaged with Chemidoc Imaging System. (H) Apparent unspliced fractions of selected introns were calculated by ratios of the intensity of the unspliced band and the sum of the unspliced band and spliced band. The intensity of bands was quantified using Fiji. Error bars report standard deviation from two biological replicates.

Article Snippet: Cells were immunostained with rabbit anti-SON antibody (1:200 dilution, Novus) or mouse anti-SRRM2 antibody (1:200 dilution, Sigma Aldrich) for 1h at room temperature followed by 3 times wash with DPBS.

Techniques: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Amplification, Electrophoresis, Immunofluorescence, Knockdown, Control, Staining, Labeling, Imaging, Standard Deviation

Journal: bioRxiv

Article Title: Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

doi: 10.1101/2024.02.29.581881

Figure Lengend Snippet: (A) IRFinder analysis showing heat shock-induced upregulation of intron retention. The number of intron retention events with more than 15% increase (ΔIR >15% ) or decrease (ΔIR <-15% ) are labeled. (B) Violin plot showing the speckle size change up heat shock compared to NT. “N” indicates the total number of speckles in each data set. (C) Viability of HeLa cells with nuclear speckle disrupted using SON/SRRM2 double knockdown (KD) or cells treated with control siRNA (siC) upon heat shock stress or NT. Hoechst staining reflected the whole cell population (with cell number denoted as N Total ), whereas Trypan blue stained the dead cell (with number denoted as N Dead ). Cell viability was calculated by 1-N Dead /N Total . P-values calculated with unpaired t-test are reported above each violin plot and box plot. Error bars report standard deviation from 3 biological replicates (in black dots). (D) 2D histogram showing the correlation between I NSE(exon) values under heat shock and NT. (E) Percentage of Group A genes and Group B and C genes without and with taking the subset of genes containing ΔIR >15% introns. P-value: Fisher’s exact test. (F) Violin plot showing the Type I sequence feature associated with three groups of introns (ΔIR >15% , ΔIR (–15%, 15%) , ΔIR <-15% ). The GC content, intron length, splice site strength and intronic ML score are compared for three groups of introns.

Article Snippet: Cells were immunostained with rabbit anti-SON antibody (1:200 dilution, Novus) or mouse anti-SRRM2 antibody (1:200 dilution, Sigma Aldrich) for 1h at room temperature followed by 3 times wash with DPBS.

Techniques: Labeling, Knockdown, Control, Staining, Standard Deviation, Sequencing

Journal: Science Advances

Article Title: Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

doi: 10.1126/sciadv.adp7727

Figure Lengend Snippet: ( A ) Scheme of ARTR-seq: Scaffold protein is immunostained by primary and secondary antibodies (Abs) sequentially. pAG-RTase then binds to the antibody to initiate reverse transcription in situ. The generated biotinylated cDNAs are collected and prepared for sequencing. ( B ) Representative image showing colocalization of Alexa Fluor 647 (AF647)–labeled pAG-RTase (magenta), Alexa Fluor 568 (AF568)–labeled secondary antibody against anti-SON primary antibody (blue), and biotinylated cDNA detected by Alexa Fluor 488 (AF488)–labeled antibody against biotin (green). The biotinylated cDNA signal in the image without the use of primary antibody (-Pri-Ab) is also shown at a fivefold lower contrast. Dashed line marks the nucleus boundary. ( C ) Two-dimensional (2D) histogram showing the correlation between I NSE determined through targeting SON and SRRM2 (in log 2 scale) in HeLa cells. Genes with lfcSE < 1 from DESeq analysis of ARTR-seq are included. Linear function ( y = ax + b ) is used to fit I NSE,SRRM2 to I NSE,SON . ( D ) Genome tracks showing ARTR-seq reads generated from targeting SON or SRRM2 and from samples without primary antibody (-Pri-Ab), mapped to MALAT1 gene. Additional genome track for -Pri-Ab is shown using autoscale for the number of reads. ( E ) RNA FISH images showing LAMA5 and P4HB transcript. RNA FISH probes were labeled with AF647 (magenta). Nuclear speckles were immunostained with AF488 (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (gray). The zoomed-in image shows one nucleus in each case. ( F ) Correlation between speckle partition coefficient ( R NS/cell ) measured by RNA FISH imaging and I NSE values determined by ARTR-seq. R NS/cell was calculated as the ratio of fluorescence signal inside nuclear speckles to that in the entire cell, which should theoretically correspond to I NSE . In (C) and (F), “ N ” reports the number of genes included in the analysis, and “ R ” reports Pearson’s correlation coefficient.

Article Snippet: Cells were immunostained with rabbit anti-SON antibody (1:200 dilution, Novus, RRID:AB_11006030), mouse anti-SRRM2 antibody (1:200 dilution, Sigma-Aldrich, RRID:AB_477511), or mouse anti-SRSF1 antibody (1:250 dilution, Invitrogen, RRID:AB_2533080) for 1 hour at room temperature followed by three-times wash with 1× PBS.

Techniques: Reverse Transcription, In Situ, Generated, Sequencing, Labeling, Staining, Imaging, Fluorescence

Journal: Science Advances

Article Title: Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

doi: 10.1126/sciadv.adp7727

Figure Lengend Snippet: ( A ) Scatter plot showing randomly selected genes from group A, B, and C genes and corresponding I NSE(exon) under NT and Plad B treatment conditions in HeLa cells. ( B ) Genome tracks showing poly (A) + RNA-seq (pink) and nuclear RNA-seq (blue) under NT and Plad B treatment conditions for selected genes: THOC6 (group A), TUBB4B (group B), and NCL (group C). Selected efficiently excised or inefficiently excised introns are highlighted in cyan and red boxes, respectively. ( C ) Schematic description of the RT-PCR assay. After reverse transcription of extracted total RNA, primers located on two adjacent exons of selected introns were used for amplification, and the PCR products were analyzed by electrophoresis. ( D ) Representative immunofluorescence images showing nuclear speckles upon SON / SRRM2 double knockdown (KD) and treated with control siRNA (siC). SON (green) and SRRM2 (magenta) were stained with AF488 and CF568 respectively. Nuclei were stained with DAPI (gray). ( E ) Histogram of SON and SRRM2 fluorescence intensity distribution under KD and siC treatment. ( F ) Violin plot showing the number of speckles per cell for KD and siC treatment. Nuclear speckles were identified by a user-defined threshold of the total intensity of the SON and SRRM2 signals, applied to both knockdown and control samples. ( G ) Representative electrophoresis analysis of RT-PCR products from THOC6 , TUBB4B , and NCL upon KD and siC treatment. ( H ) Apparent unexcised fractions of selected introns were calculated by ratios of the intensity of the unexcised band and the sum of the unexcised band and excised band. Each bar reports the mean unexcised fraction from two biological replicates. “ N ” reports the total number of cells included in each dataset in (E) and (F). P values calculated with unpaired t tests are reported above each violin plot or bar in (F) and (H).

Article Snippet: Cells were immunostained with rabbit anti-SON antibody (1:200 dilution, Novus, RRID:AB_11006030), mouse anti-SRRM2 antibody (1:200 dilution, Sigma-Aldrich, RRID:AB_477511), or mouse anti-SRSF1 antibody (1:250 dilution, Invitrogen, RRID:AB_2533080) for 1 hour at room temperature followed by three-times wash with 1× PBS.

Techniques: RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Amplification, Electrophoresis, Immunofluorescence, Knockdown, Control, Staining, Fluorescence

Journal: Science Advances

Article Title: Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

doi: 10.1126/sciadv.adp7727

Figure Lengend Snippet: ( A ) IRFinder analysis showing heat shock–induced up-regulation of intron retention. The number of intron retention events with more than 15% increase (∆IR >15% ) or decrease (∆IR <-15% ) are labeled. ( B ) Percentage of group A genes and group B and C genes without and with taking the subset of genes containing ∆IR >15% introns. P value: Fisher’s exact test. ( C ) Violin plot showing the group A–like sequence feature associated with three groups of introns (ΔIR >15% , ΔIR (−15%, 15%) , ΔIR <-15% ). The GC content, intron length, splice site strength, and intronic ML score are compared for three groups of introns. ( D ) Viability of HeLa cells under nuclear speckle disruption via SON/SRRM2 double KD or under siC upon heat shock stress or NT. Hoechst staining reflects the total cell population, whereas trypan blue stains dead cells. Cell viability was calculated as one minus the fraction of dead cells (1 − N Dead / N Total ), where N Dead and N Total are the number of dead cells and total cell number, respectively. P values calculated with unpaired t test are reported above each violin plot and box plot. Error bars report SD from three biological replicates (in black dots). ( E ) Immunofluorescence image of nuclear speckles under NT and heat shock conditions. ( F ) Violin plot showing the speckle size in heat shock compared to NT. ( G ) 2D histogram showing the correlation between I NSE(exon) values under heat shock and NT. “ N ” reports the number of introns in (A) and (C), the number of genes in (B) and (G), and the number of speckles in (F).

Article Snippet: Cells were immunostained with rabbit anti-SON antibody (1:200 dilution, Novus, RRID:AB_11006030), mouse anti-SRRM2 antibody (1:200 dilution, Sigma-Aldrich, RRID:AB_477511), or mouse anti-SRSF1 antibody (1:250 dilution, Invitrogen, RRID:AB_2533080) for 1 hour at room temperature followed by three-times wash with 1× PBS.

Techniques: Labeling, Sequencing, Disruption, Staining, Immunofluorescence