David L. Bentley

Summary: The promoter-proximal pause (PPP), at most metazoan genes, is believed to prime transcriptional activation by release of paused RNAPII into the gene. We tested the pause-release model by abolishing the PPP and monitoring gene activation. An LLM-guided AI discovered that upstream G’s and a G T/C dinucleotide robustly discriminates pauses from non-pauses. Insertion of G-less sequences into NDRG1 and HSP90AA1 abolished the PPP in an orientation-dependent way, but did not impair activation by hypoxia or heat-shock. Hence pause-release is dispensable for these responses. The 5′ peak of RNAPII detected by ChIP persisted even when pausing, detected by NET-seq was absent. Instead of pausing, these promoter-proximal polymerases may undergo abortive initiation. RNAPII C-terminal domain (CTD) deletion suppresses the PPP and shifts it upstream without affecting sequence-dependent pausing, consistent with promoter-tethering by this IDR. We propose that PPP formation is initiated by G-dependent arrest, and stabilized by multiple factors including CTD-dependent tethering.

Much pre-mRNA splicing initiates co-transcriptionally, but its mechanism is unclear. We investigated this process by degron depletion of the U1-70K subunit of U1 snRNP that contacts transcribing RNA polymerase II (RNAPII). U1-70K deficiency caused extensive exon skipping consistent with widespread exon definition. Surprisingly, exons with the strongest 5' splice sites (SSs) are skipped. Furthermore, strengthening of non-canonical 5' splice sites with a complementary mutant U1 snRNA or a splice modifying drug induced exon skipping in response to U1-70K depletion. We suggest that U1-70K plays a previously unappreciated role in destabilizing the U1-5'SS interaction to facilitate the transition to the U6-5'SS interaction required for productive splicing following exon definition. Consistent with this idea, knock- down of Prp28/DDX23 that exchanges U1 for U6 at the 5'SS also promotes exon skipping mimicking U1-70K depletion. Replacing WT U1-70K in degron-containing cells with a mutant in the RNAPII interface caused widespread exon skipping. We propose that frequent exon definition occurs at the transcription elongation complex and is enabled by U1-70K contact with transcribing RNAPII.

A signature feature of transcription on most genes in multicellular animals is that RNA polymerase II (RNAPII) piles up approximately 50 bases downstream of the start site at the promoter-proximal pause (PPP). Promoter-proximal pausing is controlled by positive and negative elongation factors that associate with RNAPII. There are two major outcomes for promoter-proximally paused RNAPII complexes: release into the gene body and premature termination. Here we discuss how RNAPII dynamics at the PPP function in a quality control checkpoint and in regulation of RNAPII flux through genes. We propose a pause release–attenuation model to describe RNAPII dynamics at the PPP.

Background: Notothenioids are a well characterised species flock endemic to the Antarctic and an important model group for the study of genome adaptation to extreme cold. We used a new reference assembly and clade-wide comparative genomic analysis to investigate cryonotothenioid evolution and the appearance of novel functionalities linked to cold adaptation. Results: A new phased assembly of a model notothenioid, Harpagifer antarcticus, demonstrated low levels of haplotypic variability across the genome. Nevertheless, numerous insertions from multiple LINE-L2 clades were found, suggesting ongoing transposition with potential contribution to speciation. Contrary to expectations the afgp locus was highly similar between haplotypes, except for large length allelic variants of afgp genes. Analysis suggests a model for the afgp locus expansion in H. antarcticus through segmental tandem duplications involving two pairs of afgp genes at time. Syntenic reconstruction of genomes from across the clade demonstrates conserved macrosyntenic relationships and group specific chromosomal fusions of notothenioids. Quantification of genome gain and transposition rates during cryonotothenioid diversification showed a first ancestral slow genome expansion concurrent with historic temperature drops. This was followed by lineage-specific massive peaks of genomic gain and transposition activity. Finally, we identified a set of genes that underwent ancestral diversifying selection and acquired novel conserved non-coding elements during the cryonotothenioid emergence. These were related to antioxidants and proteostasis, which may have facilitated the notothenioid Antarctic radiation. Conclusion: Diversifying selection and genomic gain linked to transposon activity are primary contributors to lineage-specific evolutionary dynamics through the clade which facilitated adaptation to life in the cold.

Progesterone receptors (PRs) can regulate transcription by RNA polymerase III (Pol III), which transcribes small non-coding RNAs, including all transfer RNAs (tRNAs). We previously demonstrated that PR is associated with the Pol III complex at tRNA genes and that progestins downregulate tRNA transcripts in breast tumor models. To define this mechanism, we investigated the interplay between PR, the Pol III repressor Maf1, and TFIIIB, a core transcription component. ChIP-seq was performed for PR, the Pol III subunits POLR3A, POLR3C, and POLR3G, the TFIIIB component Brf1, and Maf1 in breast cancer cells with or without progestin treatment. With progestin treatment, PR localized to ∼50% of POLR3A-occupied tRNA genes, with Maf1 co-recruited at many of these sites. Progestin treatment did not significantly alter the number of tRNA genes occupied by Pol III subunits or Brf1; however, Brf1 occupancy was stabilized. Progestins downregulated nascent transcription of one-third of tRNA genes; this repression was attenuated by Maf1 knockdown, indicating that Maf1 is required. The PR and Maf1 interaction was also detected by co-immunoprecipitation in a progestin-dependent manner. Overall, these findings demonstrate a ligand-dependent PR-mediated repression of tRNA transcription that involves Maf1.

Pre-mRNA splicing is kinetically coupled to transcription as shown by the widespread effects of transcription speed on alternative splicing (AS) outcomes. The molecular basis for such kinetic coupling is incompletely understood, but one potential mechanism is through elongation rate–dependent alternative folding pathways of the nascent pre-messenger RNA (pre-mRNA). To search for RNA structures in nascent pre-mRNA, we modified Sequencing of Psoralen Crosslinked, Ligated And Selected Hybrids (SPLASH) [J. G. Ashley Aw et al. , Mol. Cell 62 , 603–617 (2016)] for use with C hromatin A ssociated R NA. We applied this method called Chromatin Associated RNA (CAR)-SPLASH to cells expressing wild-type and slow mutant RNA polymerase II and identified >3,000 intramolecular RNA duplexes of which >400 are proximal to splice sites. Antisense oligonucleotide (ASO) disruption of several such duplexes that sequester splice sites has a major impact on AS outcomes, even though the ASOs do not directly disrupt splice sites. ASO disruption of these regulatory elements that we designate “RNA kinetic switches” modified AS of NISCH Exon 18, GAK Exon 7, and MEGF8 Exon 14 in a way that depends on the rate of transcription elongation. We propose that these switches mediate kinetic coupling via the effects of transcription speed on folding of nascent RNA structures that modulate AS and that many nascent RNA structures can thereby serve as targets for splice-modifying ASOs.

Pre-mRNA splicing is kinetically coupled to transcription as shown by the widespread effects of transcription speed on alternative splicing outcomes. The molecular basis for such kinetic coupling is incompletely understood, but one potential mechanism is through elongation rate dependent alternative folding pathways of the nascent pre-mRNA. To search for RNA structures in nascent pre-mRNA, we modified SPLASH (1) ( S equencing of P soralen Crosslinked, L igated A nd S elected H ybrids) for use with C hromatin A ssociated R NA. We applied this new method called CAR-SPLASH to cells expressing WT and slow mutant RNA polymerase II and identified > 3000 intramolecular RNA structures of which > 400 are proximal to splice sites. Antisense oligonucleotide (ASO) disruption of several such structures that sequester splice sites has a major impact on alternative splicing outcomes, even though the ASOs do not directly disrupt splice sites. ASO disruption of novel regulatory elements that we designate “RNA kinetic switches”, modified alternative splicing of NISCH Exon 18, GAK Exon 7 and MEGF8 Exon 14 in a way that depends on the rate of transcription elongation. We propose that these switches mediate kinetic coupling via effects of transcription speed on folding of nascent RNA structures that modulate alternative splicing, and that many nascent RNA structures can thereby serve as new targets for splice modifying ASOs. Significance Statement: Alternative splicing (AS) of mRNAs is a major regulator of gene expression that is frequently disrupted is disease. AS is affected by the speed of RNA polymerase II that synthesizes mRNA precursors, but how such kinetic coupling works is not well understood. We developed a method, CAR-SPLASH, to capture RNA structures in nascent RNA chains by cross-linking with psoralen. CAR-SPLASH identified novel structural regulatory elements we call “RNA kinetic switches” that control AS in a way that depends on the speed of transcription. RNA kinetic switches are new targets for splice modifying antisense oligonucleotides that could have therapeutic value.

Progesterone receptors (PR) can regulate transcription by RNA Polymerase III (Pol III), which transcribes small non-coding RNAs, including all transfer RNAs (tRNAs). We have previously demonstrated that PR is associated with the Pol III complex at tRNA genes and that progestins downregulate tRNA transcripts in breast tumor models. To further elucidate the mechanism of PR-mediated regulation of Pol III, we studied the interplay between PR, the Pol III repressor Maf1, and TFIIIB, a core transcription component. ChIP-seq was performed for PR, the Pol III subunit POLR3A, the TFIIIB component Brf1, and Maf1 in breast cancer cells with or without progestin treatment. Upon progestin exposure, PR localized to approximately half of POLR3A-occupied tRNA genes, with Maf1 co-recruited to many of these PR-POLR3A sites. While progestin treatment did not significantly alter the number of tRNA genes occupied by Pol III or Brf1, Brf1 occupancy was stabilized, as indicated by increased peak amplitudes. Analysis of nascent tRNA transcription revealed a specific progestin-induced downregulation of approximately one-third of highly expressed tRNA genes. This repression was significantly reduced by Maf1 knockdown, indicating that Maf1 is necessary for PR-mediated tRNA transcription downregulation. Overall, these findings demonstrate a ligand-dependent PR-mediated repression of tRNA transcription through Maf1.