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U1 snRNP increases RNA Pol II elongation rate to enable synthesis of long genes [RNA-Seq]

ABSTRACT: The expansion of introns within mammalian genomes poses a challenge for the production of full-length messenger RNAs (mRNA)s, with increasing evidence that these long AT-rich sequences present obstacles to transcription. Here, we investigate RNAPII elongation in mammalian cells at high resolution and demonstrate that RNAPII transcribes faster across introns. Moreover, we find that this acceleration is mediated by the association of U1 snRNP (U1) with the elongation complex at 5’ splice sites. The direct stimulation of elongation rate through introns by U1 reduces the occurrence of both premature termination and transcriptional arrest, thereby dramatically increasing RNA production. We further show that changes in RNAPII elongation rate due to AT-content and U1 binding explain previous reports of pausing or termination at splice junctions and the edge of CpG islands. We propose that this role for U1 has evolved to mitigate the risks that long, AT-rich intronic sequences pose to transcript completion.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE218125 | GEO | 2023/03/24

REPOSITORIES: GEO

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U1 snRNP increases RNA Pol II elongation rate to enable synthesis of long genes [PAC-seq]

Project description:The expansion of introns within mammalian genomes poses a challenge for the production of full-length messenger RNAs (mRNA)s, with increasing evidence that these long AT-rich sequences present obstacles to transcription. Here, we investigate RNAPII elongation in mammalian cells at high resolution and demonstrate that RNAPII transcribes faster across introns. Moreover, we find that this acceleration is mediated by the association of U1 snRNP (U1) with the elongation complex at 5’ splice sites. The direct stimulation of elongation rate through introns by U1 reduces the occurrence of both premature termination and transcriptional arrest, thereby dramatically increasing RNA production. We further show that changes in RNAPII elongation rate due to AT-content and U1 binding explain previous reports of pausing or termination at splice junctions and the edge of CpG islands. We propose that this role for U1 has evolved to mitigate the risks that long, AT-rich intronic sequences pose to transcript completion.

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U1 snRNP increases RNA Pol II elongation rate to enable synthesis of long genes [PRO-seq]

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Novel RNA-binding Proteins Bind U1 snRNA to Promote Splicing of Weak 5' Splice Sites

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Promoter directionality is controlled by U1 snRNP and polyadenylation signals

Project description:Transcription of the mammalian genome is pervasive, but productive transcription outside of protein-coding genes is limited by unknown mechanisms. In particular, although RNA polymerase II (RNAPII) initiates divergently from most active gene promoters, productive elongation occurs primarily in the sense-coding direction. Here we show in mouse embryonic stem cells that asymmetric sequence determinants flanking gene transcription start sites control promoter directionality by regulating promoter-proximal cleavage and polyadenylation. We find that upstream antisense RNAs are cleaved and polyadenylated at poly(A) sites (PASs) shortly after initiation. De novo motif analysis shows PAS signals and U1 small nuclear ribonucleoprotein (snRNP) recognition sites to be the most depleted and enriched sequences, respectively, in the sense direction relative to the upstream antisense direction. These U1 snRNP sites and PAS sites are progressively gained and lost, respectively, at the 5' end of coding genes during vertebrate evolution. Functional disruption of U1 snRNP activity results in a dramatic increase in promoter-proximal cleavage events in the sense direction with slight increases in the antisense direction. These data suggest that a U1-PAS axis characterized by low U1 snRNP recognition and a high density of PASs in the upstream antisense region reinforces promoter directionality by promoting early termination in upstream antisense regions, whereas proximal sense PAS signals are suppressed by U1 snRNP. We propose that the U1-PAS axis limits pervasive transcription throughout the genome. 3' end sequencing of poly (A) + RNAs in mouse ES cells with and without U1 snRNP inhibition using antisense morpholino oligonucleotides (AMO). Each with two biological replicates.

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Novel RNA-binding Proteins Bind U1 snRNA to Promote Splicing of Weak 5' Splice Sites

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Project description:Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative splicing. Pausing sites and changes in the rate of transcription by RNAPII may therefore have a fundamental impact in the regulation of alternative splicing. Here, we show that the elongation and splicing-related factor TCERG1 regulates alternative splicing of the apoptosis gene Bcl-x in a promoter-dependent manner. TCERG1 promotes the splicing of the short isoform of Bcl-x (Bcl-xs) through the SB1 regulatory element located in the first half of exon 2. Consistent with these results, we show evidence for in vitro and in vivo interaction of TCERG1 with the Bcl-x pre-mRNA. Transcription profile analysis reveals that the RNA sequences required for the effect of TCERG1 on Bcl-x alternative splicing coincide with a putative polymerase pause site. Furthermore, TCERG1 modifies the impact of a slow polymerase on Bcl-x alternative splicing. In support of a role for an elongation mechanism in the transcriptional control of Bcl-x alternative splicing, we found that TCERG1 modifies the amount of pre-mRNAs generated at distal regions of the endogenous Bcl-x. Most importantly, TCERG1 affects the rate of RNAPII transcription of endogenous human Bcl-x. We propose that TCERG1 modulates the elongation rate of RNAPII to relieve pausing, thereby activating the pro-apoptotic Bcl-xS 5’ splice site. ChIP-Seq

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Screening thousands of transcribed coding and non-coding regions reveals sequence determinants of RNA polymerase II elongation potential.

Project description:Organismal growth and development rely on RNA Polymerase II (RNAPII) synthesizing the appropriate repertoire of messenger RNAs (mRNAs) from protein-coding genes. Productive elongation of full-length transcripts is essential for mRNA function, however what determines whether an engaged RNAPII molecule will terminate prematurely or transcribe processively remains poorly understood. Notably, despite a common process for transcription initiation across RNAPII-synthesized RNAs1, RNAPII is highly susceptible to termination when transcribing non-coding RNAs such as upstream antisense RNAs (uaRNAs) and enhancers RNAs (eRNAs)2, suggesting that differences arise during RNAPII elongation. To investigate the impact of transcribed sequence on elongation potential, we developed a method to screen the effects of thousands of INtegrated Sequences on Expression of RNA and Translation using high-throughput sequencing (INSERT-seq). We found that higher AT content in uaRNAs and eRNAs, rather than specific sequence motifs, underlies the propensity for RNAPII termination on these transcripts. Further, we demonstrate that 5’ splice sites exert both splicing-dependent and autonomous, splicing-independent stimulation of processive transcription, even in the absence of polyadenylation signals. Together, our results reveal a potent role for transcribed sequence in dictating gene output at mRNA and non-coding RNA loci, and demonstrate the power of INSERT-seq towards illuminating these contributions.

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Widespread termination of mammalian RNA polymerase II at T-rich DNA sequences (POINT5-Seq)

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