Expression profiling by high throughput sequencing Other
Summary
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. Together, these data demonstrate that, through Sm and CHTOP arginine methylation, PRMTs regulate the post-transcriptional processing of nuclear, detained introns.
Overall design
Splicing Kinetics and Transcriptional Elongation Rates by Sequencing (SKaTER-seq) was performed two days following treatment with either 0.01% DMSO, 1 µM GSK591, or 1 µM MS023 in A549 cells. Poly(A)-RNA sequencing was completed at two-, four-, and seven-days following treatment with either 0.01% DMSO, 1 µM GSK591, or 1 µM MS023 in A549 cells.
Actinomycin D poly(A) RNA sequencing was performed two days following treatment with either 0.01% DMSO, 1 µM GSK591, or 1 µM MS023 in A549 cells. Following two days of PRMT inhibition, 5 µg/mL of Actinomycin D was added and cells were incubated for an additional 60 minutes prior to poly(A) RNA isolation and sequencing.
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