Type I and II PRMTs inversely regulate post-transcriptional intron detention through Sm and CHTOP methylation
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Evaluation Summary:
This manuscript addresses outstanding questions about the molecular mechanisms by which the two types of arginine-methylating enzymes affect the processing and fate of transcripts in mammalian cells. This work makes important inroads into these questions, uncovering an inverse effect of the two types of enzymes on intron retention during post-transcriptional splicing, linking the effects to specific target proteins. With better support of some key claims , the paper will provide a lot of new information about the functional consequences of asymmetric and symmetric demethylation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Protein arginine methyltransferases (PRMTs) are required for the regulation of RNA processing factors. Type I PRMT enzymes catalyze mono- and asymmetric dimethylation; Type II enzymes catalyze mono- and symmetric dimethylation. To understand the specific mechanisms of PRMT activity in splicing regulation, we inhibited Type I and II PRMTs and probed their transcriptomic consequences. Using the newly developed Splicing Kinetics and Transcript Elongation Rates by Sequencing (SKaTER-seq) method, analysis of co-transcriptional splicing demonstrated that PRMT inhibition resulted in altered splicing rates. Surprisingly, co-transcriptional splicing kinetics did not correlate with final changes in splicing of polyadenylated RNA. This was particularly true for retained introns (RI). By using actinomycin D to inhibit ongoing transcription, we determined that PRMTs post-transcriptionally regulate RI. Subsequent proteomic analysis of both PRMT-inhibited chromatin and chromatin-associated polyadenylated RNA identified altered binding of many proteins, including the Type I substrate, CHTOP, and the Type II substrate, SmB. Targeted mutagenesis of all methylarginine sites in SmD3, SmB, and SmD1 recapitulated splicing changes seen with Type II PRMT inhibition, without disrupting snRNP assembly. Similarly, mutagenesis of all methylarginine sites in CHTOP recapitulated the splicing changes seen with Type I PRMT inhibition. Examination of subcellular fractions further revealed that RI were enriched in the nucleoplasm and chromatin. Taken together, these data demonstrate that, through Sm and CHTOP arginine methylation, PRMTs regulate the post-transcriptional processing of nuclear, detained introns.
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Author Response:
Evaluation Summary:
This manuscript addresses outstanding questions about the molecular mechanisms by which the two types of arginine-methylating enzymes affect the processing and fate of transcripts in mammalian cells. This work makes important inroads into these questions, uncovering an inverse effect of the two types of enzymes on intron retention during post-transcriptional splicing, linking the effects to specific target proteins. With better support of some key claims , the paper will provide a lot of new information about the functional consequences of asymmetric and symmetric demethylation.
We thank the reviewers for their support of the study and hope that the described revisions better support the key claims.
Reviewer #2 (Public Review):
Previous work has established that inhibition or knockdown of the …
Author Response:
Evaluation Summary:
This manuscript addresses outstanding questions about the molecular mechanisms by which the two types of arginine-methylating enzymes affect the processing and fate of transcripts in mammalian cells. This work makes important inroads into these questions, uncovering an inverse effect of the two types of enzymes on intron retention during post-transcriptional splicing, linking the effects to specific target proteins. With better support of some key claims , the paper will provide a lot of new information about the functional consequences of asymmetric and symmetric demethylation.
We thank the reviewers for their support of the study and hope that the described revisions better support the key claims.
Reviewer #2 (Public Review):
Previous work has established that inhibition or knockdown of the Type II (symmetric) arginine methyl-transferase PRMT5 has global effects on splicing, and although it is less well-characterized, loss of the major Type I (asymmetric) enzyme PRMT1 affects both splicing and RNA export activities. In both cases, inhibition or depletion of the enzymatic activities of these proteins has been shown to negatively impact cancer cells, but the specific targets that are required for the RNA processing effects have not been identified. The key findings here are that levels of transcripts containing unspliced introns were inversely affected by the two classes of inhibitor, with intron retention increasing upon PRMT5 inhibition, and decreasing relative to the control in the case of PRMT1 inhibition. The affected introns were shown to be localized to the nucleus, indicating that they belong to the class of 'detained' introns (DI). Using kinetic assays to measure transcriptional elongation and splicing rates, the authors concluded that PRMT inhibition affects DI levels post-transcriptionally. They found that spliceosome component SNRPB and nuclear RNA export factor CHTOP were both enriched in chromatin-associated, poly(A) RNA fractions, that SNRPB was specifically demethylated by PRMT5 inhibitors while PRMT1 inhibition demethylated CHTOP in the chromatin associated fractions, and that both knockdown of the methyltransferases as well as replacement of the modified arginine residues in each protein recapitulated the effects of the inhibitors. Together, these experiments provide strong evidence supporting a coherent mechanism of differential arginine methylation on RNA processing. They support and significantly extend previously published observations implicating the PRMT enzymes in gene expression. These findings are of broad interest to those who study RNA processing and transcription, cancer biology, and signaling through post-translational modifications.
We thank the reviewer for support of our main findings and that they are of broad interest.
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Evaluation Summary:
This manuscript addresses outstanding questions about the molecular mechanisms by which the two types of arginine-methylating enzymes affect the processing and fate of transcripts in mammalian cells. This work makes important inroads into these questions, uncovering an inverse effect of the two types of enzymes on intron retention during post-transcriptional splicing, linking the effects to specific target proteins. With better support of some key claims , the paper will provide a lot of new information about the functional consequences of asymmetric and symmetric demethylation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Overall, this study provides new and compelling evidence for how methylated RBPs control RNA processing and flow through the nucleus and into the cytoplasm. One strength of the study is the use of orthogonal approaches of chemical inhibition and genetic knockdown to deplete cells of type I or type II PRMT activity. Moreover, the study presents extensive transcriptomic and proteomic data sets and analysis that will likely be useful to the community in promoting other studies. Although the manuscript does not provide a precise mechanistic investigation of how methylation controls the activity of RBPs, they do go beyond mere cataloguing of gene expression defects and link the impact of PRMTs on RNA processing to the activity of specific RBPs.
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**Reviewer #2 (Public Review):
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Previous work has established that inhibition or knockdown of the Type II (symmetric) arginine methyl-transferase PRMT5 has global effects on splicing, and although it is less well-characterized, loss of the major Type I (asymmetric) enzyme PRMT1 affects both splicing and RNA export activities. In both cases, inhibition or depletion of the enzymatic activities of these proteins has been shown to negatively impact cancer cells, but the specific targets that are required for the RNA processing effects have not been identified. The key findings here are that levels of transcripts containing unspliced introns were inversely affected by the two classes of inhibitor, with intron retention increasing upon PRMT5 inhibition, and decreasing relative to the control in the case of PRMT1 inhibition. The affected …**Reviewer #2 (Public Review):
**
Previous work has established that inhibition or knockdown of the Type II (symmetric) arginine methyl-transferase PRMT5 has global effects on splicing, and although it is less well-characterized, loss of the major Type I (asymmetric) enzyme PRMT1 affects both splicing and RNA export activities. In both cases, inhibition or depletion of the enzymatic activities of these proteins has been shown to negatively impact cancer cells, but the specific targets that are required for the RNA processing effects have not been identified. The key findings here are that levels of transcripts containing unspliced introns were inversely affected by the two classes of inhibitor, with intron retention increasing upon PRMT5 inhibition, and decreasing relative to the control in the case of PRMT1 inhibition. The affected introns were shown to be localized to the nucleus, indicating that they belong to the class of 'detained' introns (DI). Using kinetic assays to measure transcriptional elongation and splicing rates, the authors concluded that PRMT inhibition affects DI levels post-transcriptionally. They found that spliceosome component SNRPB and nuclear RNA export factor CHTOP were both enriched in chromatin-associated, poly(A) RNA fractions, that SNRPB was specifically demethylated by PRMT5 inhibitors while PRMT1 inhibition demethylated CHTOP in the chromatin associated fractions, and that both knockdown of the methyltransferases as well as replacement of the modified arginine residues in each protein recapitulated the effects of the inhibitors. Together, these experiments provide strong evidence supporting a coherent mechanism of differential arginine methylation on RNA processing. They support and significantly extend previously published observations implicating the PRMT enzymes in gene expression. These findings are of broad interest to those who study RNA processing and transcription, cancer biology, and signaling through post-translational modifications. -
Reviewer #3 (Public Review):
This manuscript focuses on addressing the contribution of two classes of arginine methyltransferases - Types I and II PRMTs - make to determining alternative splicing (AS) patterns. This question has biochemical and disease relevance because 1) the Sm proteins of snRNPs are symmetrically dimethylated by Type II (PRMT 5 and 9) enzymes, providing binding sites for SMN protein during snRNP assembly (deficiency in SMN leads to SMA), 2) many RNA binding proteins that could potentially regulate AS have arginine-rich regions that are known to be or may be modified by asymmetric or symmetric dimethylarginine (aDMA or sDMA), and 3) arginine demethylation affects biomolecular condensates that control splicing and mRNA fates.
The major strengths are the high-end sequencing strategies taken to compare transcriptomes of …
Reviewer #3 (Public Review):
This manuscript focuses on addressing the contribution of two classes of arginine methyltransferases - Types I and II PRMTs - make to determining alternative splicing (AS) patterns. This question has biochemical and disease relevance because 1) the Sm proteins of snRNPs are symmetrically dimethylated by Type II (PRMT 5 and 9) enzymes, providing binding sites for SMN protein during snRNP assembly (deficiency in SMN leads to SMA), 2) many RNA binding proteins that could potentially regulate AS have arginine-rich regions that are known to be or may be modified by asymmetric or symmetric dimethylarginine (aDMA or sDMA), and 3) arginine demethylation affects biomolecular condensates that control splicing and mRNA fates.
The major strengths are the high-end sequencing strategies taken to compare transcriptomes of cells mock treated or treated with inhibitors of the two PRMT classes; exhaustive data analyses suggest that changes in AS outcomes are due to post-transcriptional splice site choices rather co-transcriptional ones. This implies that alternative splicing choices may be predominantly post-transcriptional, an appealing idea that could help rationalize the functionalities of fast, efficient co-transcriptional splicing with slow, post-transcriptional splicing. Another strength is the isolation of proteins bound to nuclear mRNA in the presence and absence of PRMT inhibitors. The identified protein that are modified by dimethyl-arginine are candidates for mediating the effects of PRMTs.
The major weaknesses include the descriptive nature of the data, in spite of the presented evidence that dimethylated Sm and CHTOP proteins are major components accounting for the mechanistic details that must exist between the activities of Type I PRMTs (PRMT 1, 2, 3, 4, 6, 8) and Type II PRMTs. The connections between the activities of these proteins and mRNA isoforms and fates remain unclear, because post-transcriptional splicing was tested in the presence of transcription inhibition, while protein isolation was conducted by subcellular fractionation that excluded nucleoplasmic proteins. In addition, the potential roles of PRMT9 are ignored.
The authors do achieve their goal of characterizing splicing changes upon inhibition of the two classes of PRMTs. However, their conclusions regarding mechanism are limited by their preliminary nature.
The major impact for the field could be an understanding of how proteins modified by dimethylarginine bridge the gap between co-transcriptional and post-transcriptional splicing regulation. The dataset already created will be of interest to the field, as would additional datasets.
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