Productive mRNA Chromatin Escape is Promoted by PRMT5 Methylation of SNRPB
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Abstract
Protein Arginine Methyltransferase 5 (PRMT5) regulates RNA splicing and transcription by symmetric dimethylation of arginine residues (Rme2s/SDMA) in many RNA binding proteins. However, the mechanism by which PRMT5 couples splicing to transcriptional output is unknown. Here, we demonstrate that a major function of PRMT5 activity is to promote chromatin escape of a novel, large class of mRNAs that we term Genomically Retained Incompletely Processed Polyadenylated Transcripts (GRIPPs). Using nascent and total transcriptomics, spike-in controlled fractionated cell transcriptomics, and total and fractionated cell proteomics, we show that PRMT5 inhibition and knockdown of the PRMT5 SNRP (Sm protein) adapter protein pICln (CLNS1A) —but not type I PRMT inhibition—leads to gross detention of mRNA, SNRPB, and SNRPD3 proteins on chromatin. Compared to most transcripts, these chromatin-trapped polyadenylated RNA transcripts have more introns, are spliced slower, and are enriched in detained introns. Using a combination of PRMT5 inhibition and inducible isogenic wildtype and arginine-mutant SNRPB, we show that arginine methylation of these snRNPs is critical for mediating their homeostatic chromatin and RNA interactions. Overall, we conclude that a major role for PRMT5 is in controlling transcript processing and splicing completion to promote chromatin escape and subsequent nuclear export.
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Thanks for your interesting suggested experiment. We have considered a variety of reporter assays. Your proposal may be able to get at dynamics of the transport, but since most GRIPPs are also productive transcripts, may not on its own have adequate dynamic range to test mechanisms. We will think about this further.
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Thanks for your interesting question. We show in Figure 6h,i that there are transcripts pre-disposed to be found on chromatin. Figure 7f also shows that in control cells, there are many transcripts pre-disposed to contain intronic reads. We suspect that GRIPPs may indeed act as a sensor for some stressed conditions, for which PRMT5 is a master regulator, and are currently further testing this question.
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Thanks for your interesting question. Our evidence supports a model in which generally slower splicing leads to intron detention across GRIPP transcripts. We have no evidence of a specific identification mechanism. In Supplemental Fig S6d-g, we show that there is no specific enrichment of intronic reads. The physical association with chromatin we hypothesize may be due to altered condensate formation between polyanions (RNA and DNA) and the unmethylated SNRP IDRs, or alternatively through reduced rate of U5 snRNP turnover and release.
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Lastly, while we have characterized GRIPPs as a novel transcript class, further studies will explore their genomic characteristics and how they are protected from RNA degradation by the RNA exosome complex and prevented from RNA nuclear export.
To identify genetic regulators of GRIPP processing and export, have you considered a functional genomic approach? If you were able to integrate a reporter into a GRIPP, such as a GFP, that would only be expressed when the GRIPP was processed and exported to the cytosol, you might be able to use that to identify genetic regulators of this process in the presence and absence of PRMT5 inhibition.
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Strikingly, we found that if a transcript has zero introns, it is unlikely to be either regulated by PRMT5 inhibition or trapped on chromatin. In contrast, if a transcript has one or more introns, it is more likely to be both regulated by and enriched on chromatin upon PRMT5 inhibition
The proposed mechanism that the presence of introns leads to increased retention of certain transcripts on chromatin is very intriguing. How does this system identify introns in a transcript, and how does this lead to physical association with chromatin? Also, do you know why only certain intron-containing transcripts (i.e. those associated with RNA processing) are retained, and not others?
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Comparing 48-hour GSK591- and DMSO treated chromatin proteomes revealed gene ontology alteration of proteins related to RNA processing, RNA binding, and splicing (Figure 3e-f). This is consistent with the transcriptomic ontology analysis at similar two days of PRMT5 inhibition
Do the transcriptomic and proteomic effects of PRMT5 inhibition on RNA processing represent a pre-encoded cellular response for which PRMT5 serves as a master regulator? What kind of cellular conditions (outside of inhibition or KD, i.e. those that might arise in the environment) would lead to activation of this process?
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