Biology subjects

Gamble, M. J.

Publications and source records attributed to Gamble, M. J..

3 recordsLinked to original sources

bioRxiv2021

Type I PRMTs and PRMT5 Inversely Regulate Post-Transcriptional Intron Detention

Protein arginine methyltransferases (PRMTs) are required for the regulation of RNA processing factors. Type I 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 SKaTER-seq method, analysis of co-transcriptional splicing revealed that PRMT inhibition resulted in slower splicing rates. Surprisingly, altered co-transcriptional splicing kinetics correlated poorly with ultimate changes in alternative splicing of polyadenylated RNA--particularly intron retention (RI). Investigation of RI following inhibition of nascent transcription demonstrated that PRMTs inversely regulate RI post-transcriptionally. Subsequent proteomic analysis of chromatin-associated polyadenylated RNA identified aberrant binding of 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. Conversely, mutagenesis of all methylarginine sites in CHTOP recapitulated the splicing changes seen with Type I PRMT inhibition. Closer examination of subcellular fractions indicated that RI were isolated to the nucleoplasm and chromatin. Together, these data demonstrate that PRMTs regulate the post-transcriptional processing of nuclear, detained introns through Sm and CHTOP arginine methylation.

cancer biology↗

bioRxiv2021

Spatial organization of transcript elongation and splicing kinetics

The organization of the genome in three-dimensional space has been shown to play an important role in gene expression. Specifically, facets of genomic interaction such as topologically associated domains (TADs) have been shown to regulate transcription by bringing regulatory elements into close proximity1. mRNA production is an intricate process with multiple control points including regulation of Pol II elongation and the removal of non-coding sequences via pre-mRNA splicing2. The connection between genomic compartments and the kinetics of RNA biogenesis and processing has been largely unexplored. Here, we measure Pol II elongation and splicing kinetics genome-wide using a novel technique that couples nascent RNA-seq with a mathematical model of transcription and co-transcriptional RNA processing. We uncovered multiple layers of spatial organization of these rates: the rate of splicing is coordinated across introns within individual genes, and both elongation and splicing rates are coordinated within TADs, as are alternative splicing outcomes. Overall, our work establishes that the kinetics of transcription and splicing are coordinated by the spatial organization of the genome and suggests that TADs are a major platform for coordination of alternative splicing.

molecular biology↗

bioRxiv2020

Type I PRMTs and PRMT5 Independently Regulate Both snRNP Arginine Methylation and Post-Transcriptional Splicing

Protein arginine methyltransferases (PRMTs) methylate histones, splicing factors, and many other nuclear proteins. Type I enzymes (PRMT1-4,6,8) catalyze mono- (Rme1/MMA) and asymmetric (Rme2a/ADMA) dimethylation; Type II enzymes (PRMT5,9) catalyze mono- and symmetric (Rme2s/SDMA) dimethylation. Misregulation of PRMTs in multiple types of cancers is associated with aberrant gene expression and RNA splicing. To understand the specific mechanisms of PRMT activity in splicing regulation, we treated cells with the PRMT5 inhibitor GSK591 and the Type I inhibitor MS023 and probed their transcriptomic consequences. We discovered that Type I PRMTs and PRMT5 inversely regulate core spliceosomal Sm protein Rme2s and intron retention. Loss of Sm Rme2s is associated with the accumulation of polyadenylated RNA containing retained introns and snRNPs on chromatin. Conversely, increased Sm Rme2s correlates with decreased intron retention and chromatin-association of intron-containing polyadenylated RNA. Using the newly developed SKaTER-seq model, comprehensive and quantitative analysis of co-transcriptional splicing revealed that either Type I PRMT or PRMT5 inhibition resulted in slower splicing rates. Surprisingly, altered co-transcriptional splicing kinetics correlated poorly with ultimate changes in alternatively spliced mRNA. Quantitation of retained intron decay following inhibition of nascent transcription revealed that Type I PRMTs and PRMT5 reciprocally regulate post-transcriptional splicing efficiency.

cell biology↗