Spatial organization of transcript elongation and splicing kinetics

Casill, Alyssa D.; Haimowitz, Adam J.; Kosmyna, Brian; Query, Charles C.; Ye, Kenny; Gamble, Matthew J.

doi:10.1101/2021.01.28.428713

Cited by 5 publications

(11 citation statements)

References 47 publications

(77 reference statements)

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“…As predicted, we observed a strongly significant (P < 2.2e-16) correlation between RNA pol II spawn rate and TPM in DMSO-, GSK591-, and MS023-treated cells (ρ = 0.50, 0.51, 0.51, respectively) (Suppl. Figure 3a) (Casill et al 2021). Consistent with previously published reports, a comparison of splicing rates within each condition confirmed that constitutive introns splice faster than cassette exons as well as alternative 5' and 3' splice sites (Suppl.…”

Section: Prmt-dependent Changes In Co-transcriptional Splicing Do Not Reflect Changes In Mrnasupporting

confidence: 91%

“…The inverse relationship on RI by Type I or PRMT5i in addition to their TES-proximal location, shorter length, and weaker splice sites led us to investigate the kinetics of co-transcriptional splicing. To accomplish this, we used Splicing Kinetics and Transcript Elongation Rates by Sequencing (SKaTER seq) (Casill et al 2021). As splicing changes are present as early as two days following GSK591 or MS023 treatment, we used this time point of PRMTi for our analysis.…”

Section: Prmt-dependent Changes In Co-transcriptional Splicing Do Not Reflect Changes In Mrnamentioning

confidence: 99%

“…Nascent RNA isolated via a 1 M urea wash of chromatin and an additional poly(A)-RNA depletion is then sequenced. The rate of nascent RNA formation-including: (1) RNA pol II initiation and pauserelease (spawn) rate, (2) elongation rate, (3) splicing rate, and (4) transcript cleavage rate-is then calculated by using a comprehensive model that determines the rates that best fit the sequencing coverage (Casill et al 2021).…”

Section: Prmt-dependent Changes In Co-transcriptional Splicing Do Not Reflect Changes In Mrnamentioning

confidence: 99%

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

Maron¹,

Casill

Gupta³

et al. 2021

Preprint

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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.

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Section: Prmt-dependent Changes In Co-transcriptional Splicing Do Not Reflect Changes In Mrnasupporting

confidence: 91%

Section: Prmt-dependent Changes In Co-transcriptional Splicing Do Not Reflect Changes In Mrnamentioning

confidence: 99%

Section: Prmt-dependent Changes In Co-transcriptional Splicing Do Not Reflect Changes In Mrnamentioning

confidence: 99%

See 1 more Smart Citation

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

Maron¹,

Casill

Gupta³

et al. 2021

Preprint

Self Cite

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show abstract

“…The inverse relationship on RI by Type I or II inhibition in addition to their TES-proximal location, shorter length, and non-canonical splice sites led us to specifically investigate the kinetics of co-transcriptional splicing. To accomplish this, we used Splicing Kinetics and Transcript Elongation Rates by Sequencing (SKaTER seq) ( Casill et al, 2021 ). A brief overview of the method follows.…”

Section: Resultsmentioning

confidence: 99%

“…(2) elongation rate, (3) splicing rate, and (4) transcript cleavage rate-is calculated using a comprehensive model that determines the rates that best fit the sequencing coverage (Casill et al 2021).…”

Section: Prmt-dependent Changes In Co-transcriptional Splicing Do Not Reflect Changes In Poly(a) Rnamentioning

confidence: 99%

Type I and II PRMTs inversely regulate post-transcriptional intron detention through Sm and CHTOP methylation

Maron

Casill

Gupta

et al. 2022

eLife

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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.

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Role of Dot1L and H3K79 methylation in regulating somatic hypermutation of immunoglobulin genes

Duan

Baughn

Wang

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Somatic hypermutation (SHM) and class-switch recombination (CSR) of the immunoglobulin (Ig) genes allow B cells to make antibodies that protect us against a wide variety of pathogens. SHM is mediated by activation-induced deaminase (AID), occurs at a million times higher frequency than other mutations in the mammalian genome, and is largely restricted to the variable (V) and switch (S) regions of Ig genes. Using the Ramos human Burkitt’s lymphoma cell line, we find that H3K79me2/3 and its methyltransferase Dot1L are more abundant on the V region than on the constant (C) region, which does not undergo mutation. In primary naïve mouse B cells examined ex vivo, the H3K79me2/3 modification appears constitutively in the donor Sμ and is inducible in the recipient Sγ1 upon CSR stimulation. Knockout and inhibition of Dot1L in Ramos cells significantly reduces V region mutation and the abundance of H3K79me2/3 on the V region and is associated with a decrease of polymerase II (Pol II) and its S2 phosphorylated form at the IgH locus. Knockout of Dot1L also decreases the abundance of BRD4 and CDK9 (a subunit of the P-TEFb complex) on the V region, and this is accompanied by decreased nascent transcripts throughout the IgH gene. Treatment with JQ1 (inhibitor of BRD4) or DRB (inhibitor of CDK9) decreases SHM and the abundance of Pol II S2P at the IgH locus. Since all these factors play a role in transcription elongation, our studies reinforce the idea that the chromatin context and dynamics of transcription are critical for SHM.

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Spatial organization of transcript elongation and splicing kinetics

Cited by 5 publications

References 47 publications

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

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

Type I and II PRMTs inversely regulate post-transcriptional intron detention through Sm and CHTOP methylation

Role of Dot1L and H3K79 methylation in regulating somatic hypermutation of immunoglobulin genes

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