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Transcription and splicing: A two‐way street

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Abstract RNA synthesis by RNA polymerase II and RNA processing are closely coupled during the transcription cycle of protein‐coding genes. This coupling affords opportunities for quality control and regulation of gene expression and the effects can go in both directions. For example, polymerase speed can affect splice site selection and splicing can increase transcription and affect the chromatin landscape. Here we review the many ways that transcription and splicing influence one another, including how splicing "talks back" to transcription. We will also place the connections between transcription and splicing in the context of other RNA processing events that define the exons that will make up the final mRNA. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing
The splicing cycle. (a) Splicing is a two‐step transesterification reaction. In the first step, the 5′SS undergoes cleavage via a nucleophilic attack from the 2′OH group of the branchpoint (BP) adenine. The 5′ splice site (SS) becomes covalently linked to the BP, forming the lariat intermediate. In the second step, the 3′OH from the 5′ exon attacks the 3′SS, resulting in the ligation of the 5′ and 3′ exons and the release of the intron. (b) The spliceosome cycle. The 5 snRNPs that constitute the spliceosome are thought to assemble on the pre‐mRNA transcript in a step‐wise manner. First, the U1 snRNP binds to the 5′SS and the branchpoint is bound by branchpoint binding protein (forming the E complex), followed by the U2 snRNP which binds to the BP (forming the A complex). The U4/U6•U5 tri‐snRNP then joins forming the pre‐B complex. U1 leaves and the pre‐catalytically active spliceosome (B complex). The spliceosome undergoes substantial re‐arrangements between RNA and protein interactions to produce the active spliceosome (Bact‐> B* complex) that catalyzes the first step of splicing. The C complex is then formed and converted to C* complex that catalyzes the second step of splicing (exon ligation) to generate a mature RNA transcript with post‐spliceosome (P complex). The spliceosome is disassembled and lariat released. Source: Fica & Nagai, ; Will & Luhrmann,
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Definition of the first and last exons in humans. (a) Definition of the first exon. This involves binding of the RNA 5′ end by the cap‐binding complex (CBC), followed by interaction between the CBC and the U4/U6·U5 tri‐snRNP. The CBC also mediates the definition of the 3′ end of the first exon through the binding of the U1 snRNP to the initial 5′ splice site. The U4/U6·U5 tri‐snRNP will later on replace the U1 snRNP during spliceosome maturation and catalysis. (b) Definition of the last exon. This involves recognition of the final 3′ splice site by the U2 snRNP just upstream of the 5′ end of the exon, and recognition of the final poly(A) signal by the CPA complex at the 3′ end of the exon. Direct interactions between the U2 snRNP, the CPSF complex and the poly(A) polymerase mediate the definition of the last exon. Interactions between elongation factors, such as CDC73, which is part of the PAF1 complex, and CPSF and CstF proteins may help to simultaneously define the last exon, promote mRNA cleavage and polyadenylation, and effect pol II termination
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Splicing talks back to transcription and chromatin. (a) Several splicing and splicing‐related factors are involved in the regulation of pol II pausing downstream of the TSS. The splicing factor, SRSF2, and the pre‐exon‐junction complex (pre‐EJC) could compete for binding to the nascent RNA. Whereas SRSF2 binding stimulates pause release and therefore transcription elongation through the recruitment of active P‐TEFb to the paused pol II, the pre‐EJC helps keep pol II paused. The pre‐EJC has also been found to regulate, most likely indirectly, nucleosome and H3K4me3 levels. Inhibition of SF3b1 by the splicing inhibitor, Spliceostatin A (SSA), also causes H3K4me3 levels to decrease but it is unknown if this is direct or indirect. In addition, pol II pausing is affected by knockdown of cleavage and polyadenylation (CPA) factors, possibly due to a decrease in pol II premature termination. (b) Elongating pol II is also regulated by splicing. Usage of intronic poly(A) sites, which if used would result in truncated mRNAs, is blocked by the presence of the U1 snRNP, which inhibit the recognition of the intronic poly(A) site by the CPA complex through interactions with CPA factors. In yeast, co‐transcriptional splicing induces pol II pausing at the end of introns to provide enough time for splicing to occur. Several splicing inhibitors cause a loss of H3K36me3, a decrease in pol II and pol II CTD phosphorylation levels, and changes in alternative splicing. (c) Inhibition of splicing affects pol II termination. At the end of protein‐coding genes, inhibition of splicing by Isoginkgetin or SSA promotes termination defects resulting in pol II elongating downstream of the poly(A) site beyond the normal termination site. Termination defects can also be induced by knockdown of CPA factors or with cellular stress, such as viral infection or osmotic stress
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Kinetic coupling. The pol II elongation rate affects co‐transcriptional spliceosome assembly and splice site selection. The kinetic model proposes that when pol II elongation is slow, weak splice sites are favored and alternative exons with weak splice sites are included. When pol II elongation is fast, strong splice sites are favored and alternative exons with weak splice sites are skipped. However, this is not always the case (see text)
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The splicing and transcription cycles are coupled in space and time. During co‐transcriptional splicing, the spliceosome assembles on the nascent pre‐mRNA. In this way, splicing, transcription and chromatin are in close‐proximity and influence each other. The CTD of pol II (YSPTSPS) and pol II ‐associated elongation complex (EC) are shown. In this and subsequent figures, S2 and S5 CTD phosphorylation are highlighted by red “P,” the spliceosome and its interactions are shown by the blue circle with dashed lines, and nucleosomes are shown as barrels with post‐translational modifications of histones shown as colored stars
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RNA Processing > Splicing Mechanisms

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