Type I and II PRMTs inversely regulate post-transcriptional intron detention through Sm and CHTOP methylation
- Department of Biochemistry, Albert Einstein College of Medicine, United States
- Department of Molecular Pharmacology, Albert Einstein College of Medicine, United States
- Department of Cell Biology, Albert Einstein College of Medicine, United States
Figures
Figure 1 with 2 supplements
Type I PRMTs and PRMT5 inversely regulate intron retention.
(a) Overview of protein arginine methyltransferases and their catalyzed reactions. (b) Comparison of ΔΨ for retained introns (RI) following PRMT inhibition where ΔΨ=Ψ (PRMT inhibitor)–Ψ (DMSO). …
Figure 1—figure supplement 1
PRMTkd recapitulates RI inclusion seen with PRMT inhibition.
(a, b) RT-qPCR of PRMT expression (a) or RI (b) 4 days after transduction of A549 cells with gRNAs targeting the indicated PRMT. Data are represented as mean ± SD. Significance determined using …
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Figure 1—figure supplement 1—source data 1
Western blot data for Figure 1—figure supplement 1c.
- https://cdn.elifesciences.org/articles/72867/elife-72867-fig1-figsupp1-data1-v2.zip
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Figure 1—figure supplement 1—source data 2
Western blot data for Figure 1—figure supplement 1d.
- https://cdn.elifesciences.org/articles/72867/elife-72867-fig1-figsupp1-data2-v2.zip
Figure 1—figure supplement 2
PRMTs regulate a conserved class of retained introns (RI) that share unique features.
(a) Matrix comparing the log2(odds ratio) and significance as determined by the Fisher’s exact test of overlapping RI from indicated experimental models. (b) Web logo diagram of nucleotide …
Figure 2 with 2 supplements
Retained introns (RI) share unique characteristics and are independent of PRMT-regulated co-transcriptional splicing.
(a) Histogram of SKaTER-seq aligned reads across WASL. (b) Distribution of splicing rates for common genomic introns. Dashed line indicates DMSO median; solid line within boxplot is …
Figure 2—figure supplement 1
Transcript spawn rate correlates with expression and alternative splicing events are slower than constitutive ones.
(a) Correlation of SKaTER-seq model simulated cassette exon Ψ versus poly(A)-RNA seq Ψ. (b) Correlation of RNA pol II initiation and pause-release (spawn rate) with poly(A)-RNA seq transcripts per …
Figure 2—figure supplement 2
Splicing rate and probability of cleavage prior to splicing correlate with intron position.
(a) Correlation of splicing rate and intron position in cells treated with DMSO, GSK591, or MS023 for 2 days. (b) Probability of cleavage prior to splicing for common genome-wide introns relative to …
Figure 3
Type I and II PRMTs regulate RI post-transcriptionally.
(a) Overview of Actinomycin D (ActD) poly(A)-RNA sequencing protocol. (b) Genome browser track of poly(A)-RNA seq aligned reads (−) and (+) ActD for NOP2. (c) Distribution of log2(+ActD/−ActD) …
Figure 4 with 1 supplement
PRMT inhibition promotes aberrant binding of RNA processing factors to chromatin-associated poly(A) RNA.
(a) Overview of chromatin-associated poly(A)-RNA LC-MS/MS experiment. (b, c) Dot plot of proteins bound to chromatin (b) or chromatin-associated poly(A) RNA (c) relative to DMSO. Circle size is …
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Figure 4—source data 1
Western blot data for Figure 4f.
- https://cdn.elifesciences.org/articles/72867/elife-72867-fig4-data1-v2.zip
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Figure 4—source data 2
Western blot data for Figure 4g.
- https://cdn.elifesciences.org/articles/72867/elife-72867-fig4-data2-v2.zip
Figure 4—figure supplement 1
PRMT inhibition alters association of RNA splicing, localization, and processing factors to chromatin and chromatin-associated poly(A) RNA.
(a, b) Enriched biological processes found in the proteome of input chromatin (a) and chromatin-associated poly(A) RNA (b) in A549 cells treated with GSK591 or MS023 relative to DMSO.
Figure 5 with 2 supplements
Sm and CHTOP methylarginine mediates PRMT-dependent RI.
(a) Comparison of ΔΨ for RI following SNRPB knockdown (kd), GSK591 treatment, MS023 treatment, and CHTOPkd where ΔΨ=Ψ (Treatment)–Ψ (Control). (b, c) Comparison of ΔΨ z-score for common RI between …
Figure 5—figure supplement 1
CHTOP and Sm arginine mutants phenocopy PRMT inhibition.
(a) Comparison of ΔΨ for RI following ALYREFkd and CHTOPkd where ΔΨ=Ψ (Treatment)–Ψ (Control). (b) RT-qPCR of Exogenous Sm R-to-A and R-to-K mutants normalized to wild-type (WT) expression vector. …
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Figure 5—figure supplement 1—source data 1
Western blot data for Figure 5—figure supplement 1c.
- https://cdn.elifesciences.org/articles/72867/elife-72867-fig5-figsupp1-data1-v2.zip
Figure 5—figure supplement 2
Sm methylarginine is dispensable for snRNP assembly.
(a) Western blot analysis of FLAG-targeted immunoprecipitation in A549 cells expressing Sm wild-type (WT), R-to-A, or R-to-K mutants. DB71=Direct Blue 71 membrane stain. See Figure 5—figure …
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Figure 5—figure supplement 2—source data 1
Western blot data for Figure 5—figure supplement 2a.
- https://cdn.elifesciences.org/articles/72867/elife-72867-fig5-figsupp2-data1-v2.zip
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Figure 5—figure supplement 2—source data 2
Northern blot data for Figure 5—figure supplement 2b.
- https://cdn.elifesciences.org/articles/72867/elife-72867-fig5-figsupp2-data2-v2.zip
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Figure 5—figure supplement 2—source data 3
Northern blot data for Figure 5—figure supplement 2c.
- https://cdn.elifesciences.org/articles/72867/elife-72867-fig5-figsupp2-data3-v2.zip
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Figure 5—figure supplement 2—source data 4
Image data for Figure 5—figure supplement 2d.
- https://cdn.elifesciences.org/articles/72867/elife-72867-fig5-figsupp2-data4-v2.zip
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Figure 5—figure supplement 2—source data 5
Image data for Figure 5—figure supplement 2e.
- https://cdn.elifesciences.org/articles/72867/elife-72867-fig5-figsupp2-data5-v2.zip
Figure 6
PRMT-dependent RI are localized to the nucleoplasm and chromatin.
(a) Scatter plot of ΔΨ for common RI in A549 nuclear/cytoplasmic fractions and GSK591 (left, green) or MS023 (right, purple) treated cells where ΔΨ=Ψ (Nuclear/PRMT inhibition)–Ψ (Cytoplasm/DMSO). (b)…
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Figure 6—source data 1
Western blot data for Figure 6c.
- https://cdn.elifesciences.org/articles/72867/elife-72867-fig6-data1-v2.zip
Figure 7
Type I and II PRMTs regulate post-transcriptional intron detention through Sm and CHTOP methylation.
(a–c) Model figures representing an overview of Type I- and Type II-dependent regulation of detained introns (DI) (a). Sm Rme2s is necessary for productive splicing and maturation of DI; absence of …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | A549 | ATCC | Cat#: CCL-185RRID:CVCL_0023 | |
| Transfected construct (H. sapiens) | Wild-type FLAG-SmD3-p2a-FLAG-SmB-p2a-V5-SmD1 | VectorBuilder | VB210103-1026dg | Lentiviral construct to transfect and express the wild-type Sm proteins. |
| Transfected construct (H. sapiens) | R-to-A FLAG-SmD3-p2a-FLAG-SmB-p2a-V5-SmD1 | VectorBuilder | VB210103-1027ttz | Lentiviral construct to transfect and express the R-to-A mutant Sm proteins. |
| Transfected construct (H. sapiens) | R-to-K FLAG-SmD3-p2a-FLAG-SmB-p2a-V5-SmD1 | VectorBuilder | VB210317-1185yxr | Lentiviral construct to transfect and express the R-to-K mutant Sm proteins. |
| Transfected construct (H. sapiens) | Wild-type FLAG-CHTOP | VectorBuilder | VB210427-1238rhx | Lentiviral construct to transfect and express the wild-type CHTOP protein. |
| Transfected construct (H. sapiens) | R-to-A FLAG-CHTOP | VectorBuilder | VB210427-1241jrw | Lentiviral construct to transfect and express the R-to-A CHTOP protein. |
| Transfected construct (H. sapiens) | R-to-K FLAG-CHTOP | VectorBuilder | VB210427-1242gjh | Lentiviral construct to transfect and express the R-to-K CHTOP protein. |
| Transfected construct (H. sapiens) | dCas9-KRAB-MeCP2 | VectorBuilder | VB900120-5303pyt | Lentiviral construct to transfect and express dCas9-KRAB-MeCP2. |
| Transfected construct (H. sapiens) | gRNA parent vector | VectorBuilder | VB210119-1169qwd | Lentiviral construct to clone, transfect, and expressPRMTkd gRNAs (see Materials and methods). |
| Antibody | Anti-human SNRPB (Rabbit polyclonal) | ProteinTech | Cat#: 16807-1-AP; RRID:AB_2878319 | WB: 1:2000IF: 1:125IP: 5 μg |
| Antibody | Anti-FLAG (Mouse monoclonal) | Sigma-Aldrich | Cat#: F1804; RRID:AB_262044 | WB: 1:10,000IF: 1:50IP: 5 μg |
| Antibody | Anti-Rabbit IgG (Goat polyclonal) | Thermo Fisher Scientific | Cat#: 35552; RRID:AB_844398 | IF: 1:1000 |
| Antibody | Anti-Mouse IgG (Goat polyclonal) | Thermo Fisher Scientific | Cat#: A28180RRID:AB_2536164 | IF: 1:1000 |
| Antibody | Anti-Rme2s (Rabbit polyclonal) | CST | Cat#: 13222SRRID:AB_2714013 | WB: 1:2000 |
| Antibody | Anti-Rme2a (Rabbit polyclonal) | CST | Cat#: 13522SRRID:AB_2665370 | WB: 1:2000 |
| Antibody | Anti-Rme1 (Rabbit polyclonal) | CST | Cat#: 8015SRRID:AB_10891776 | WB: 1:2000 |
| Antibody | Anti-Mouse CHTOP (Rat monoclonal) | LSBio | Cat#: LS-B11259-50 | WB: 1:2000IP: 5 μg |
| Antibody | Anti-human SNRPD3(Rabbit polyclonal) | Abcam | Cat#: ab157118 | WB: 1:2000 |
| Antibody | Anti-human LaminB2 (Mouse monoclonal) | Thermo Fisher Scientific | Cat#: MA1-06104RRID:AB_2136415 | WB: 1:2000 |
| Antibody | Anti-human U1-70k (Mouse monoclonal) | Santa Cruz Biotechnology | Cat#: sc-390988 | WB: 1:2000 |
| Antibody | Anti-human H3 (Rabbit polyclonal) | Abcam | Cat#: ab1791RRID:AB_302613 | WB: 1:100,000 |
| Antibody | Anti-human GAPDH (Mouse monoclonal) | Abcam | Cat#: ab9484RRID:AB_307274 | WB: 1:10,000 |
| Antibody | Anti-human PRMT1 (Rabbit polyclonal) | Millipore | Cat#: 07-404RRID:AB_11212188 | WB: 1:2000 |
| Antibody | Anti-human PRMT5 (Rabbit polyclonal) | Millipore | Cat#: 07-405RRID:AB_310589 | WB: 1:2000 |
| Antibody | Anti-Rat IgG HRP (Goat polyclonal) | Millipore | Cat#: AP183PMI | WB: 1:100,000 |
| Antibody | Anti-Rabbit IgG HRP (Goat polyclonal) | Cytiva | Cat#: NA934 | WB: 1:100,000 |
| Antibody | Anti-Mouse IgG HRP (Goat polyclonal) | Cytiva | Cat#: NA931 | WB: 1:100,000 |
| Chemical compound, drug | DMSO | Acros Organics | Cat#: D/4125/PB08 | |
| Chemical compound, drug | GSK591 | Cayman | Cat#: 18354 | |
| Chemical compound, drug | MS023 | Cayman | Cat#: 18361 | |
| Chemical compound, drug | Actinomycin D | Sigma-Aldrich | Cat#: A1410 | |
| Software, algorithm | STAR | Dobin et al., 2013 | RRID:SCR_004463 | Version 2.4.2 |
| Software, algorithm | rMATS | Shen et al., 2014 | RRID:SCR_013049 | Version 4.1.0 |
| Software, algorithm | Kallisto | Bray et al., 2016 | RRID:SCR_016582 | Version 0.46.0 |
| Software, algorithm | Proteome Discoverer software | Thermo Fisher Scientific | Version 2.4 | |
| Software, algorithm | Prism | Graphpad | RRID:SCR_002798 | |
| Software, algorithm | R | R Project | RRID:SCR_001905 | Version 4.0.2 |
| Other | DAPI Prolong gold | Invitrogen | Cat#: P36941 |
Additional files
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Supplementary file 1
Chromatin-Associated Poly(A) RNA Enrichment.
Enrichment and relative abundance of proteins in chromatin or chromatin-associated poly(A) RNA following treatment with DMSO, GSK591, or MS023 for two days.
- https://cdn.elifesciences.org/articles/72867/elife-72867-supp1-v2.xlsx
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Supplementary file 2
Oligonucleotide sequences used in this study.
- https://cdn.elifesciences.org/articles/72867/elife-72867-supp2-v2.xlsx
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Supplementary file 3
Publicly available data used in this study.
- https://cdn.elifesciences.org/articles/72867/elife-72867-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/72867/elife-72867-transrepform1-v2.docx
