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Pre‐mRNA splicing during transcription in the mammalian system

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Abstract Splicing of RNA polymerase II transcripts is a crucial step in gene expression and a key generator of mRNA diversity. Splicing and transcription have generally been studied in isolation, although in vivo pre‐mRNA splicing occurs in concert with transcription. The two processes appear to be functionally connected because a number of variables that regulate transcription have been identified as also influencing splicing. However, the mechanisms that couple the two processes are largely unknown. This review highlights the observations that implicate splicing as occurring during transcription and describes the evidence supporting functional interactions between the two processes. I discuss postulated models of how splicing couples to transcription and consider the potential impact that such coupling might have on exon recognition. WIREs RNA 2011 2 700–717 DOI: 10.1002/wrna.86 This article is categorized under: RNA Processing > Capping and 5' End Modifications RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing RNA Processing > 3' End Processing

Endogenous pre‐mRNA's assemble ‘exon defined’ rather than ‘intron defined’ complexes. (a) In metazoans, where introns are much longer than exons, the U1 snRNP bound at a 5′ss stimulates binding of U2AF at the upstream 3′ss to form a cross‐exon E complex. In this manner, each exon is recognized as an independent unit prior to pairing of two exons across an intron, and is known as ‘exon definition’. (b) RNAs containing short introns, such as those used for in vitro splicing, assemble spliceosomes via intron definition. Under intron‐defined conditions, U1 bound at a 5′ss will form a cross intron E complex with U2AF bound at the downstream 3′ss. (c) A prediction of the exon definition model is that transcription of the entire alternative region be complete prior to splicing. Under these conditions, the stronger, more rapidly assembling 3′ splice site of the downstream constitutive exon is predicated to outcompete the weaker 3′ss of the alternative exon for pairing with the upstream constitutive exon. This would result in regulated exon skipping. (d) Under conditions of slower transcriptional elongation or polymerase pausing in the downstream intron, alternative exon definition and pairing can proceed without competition from the stronger constitutive downstream exon, thus providing a potential mechanism by which enhanced inclusion of the regulated exon can be achieved while still adhering to an exon‐defined mode of splicing.

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Alternative splicing choices are affected by transcriptional‐ and chromatin‐associated parameters. The boxed regions and arrowheads represent the different aspects of chromatin structure and transcriptional dynamics that are known to affect splicing choices. The annotation(s) next to each arrowhead indicate the reference number for the studies that describe each effect. (a) FN alternative exon 33 inclusion levels differ when transcribed from different promoter sequences. (b) Transcriptional activators that bind to promoter sequences, such as the CAPER proteins, can alter the splicing patterns of mRNAs produced from the downstream transcriptional unit. It is not known whether this effect is direct or through changes in the rate of transcription. (c) Nucleosome remodeling factors can influence regulated splicing choices. The SWI/SNF subunit Brm1 can bind to the U1 and U5 snRNPs and alter splicing patters of RNAs transcribed from genes that require SWI/SNF remodeling at the promoter for activation. (d) Dense chromatin structures are predicted to hinder polII accessibility to the DNA and possibly slow transcriptional rates. The kinetic model posits that this will increase alternative exon inclusion rates. Conversely, relaxed chromatin is thought to enhance transcription and thus, favor regulated exon skipping. (e) SiRNA's targeted to intronic regions upstream of alternative FN exon 33 induce chromatin condensation and increase inclusion of FN exon 33. Depolarizing stimuli leads to a more open chromatin state and a decrease in inclusion of the NCAM exon 18. (f) Chromatin structure is implicated in regulating transcriptional rate, which in turn might effect changes in alternative splicing choices as posited by the kinetic model. Alternatively, some of these variables may interfere with spliceosome assembly to influence splicing choice.

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A slower rate of transcriptional elongation promotes alternative exon inclusion. (a) Polymerases working at a relatively fast rate of transcription will rapidly transcribe the alternative region, such that the 3′ splice site of the alternative and downstream constitutive exons are both available for pairing with the 5′ss of the upstream constitutive exon. Competition between the stronger constitutive and weaker alternative 3′ splice sites will favor use of the downstream constitutive 3′ss and promote alternative exon skipping. (b) The kinetic model posits that a slower transcriptional elongation rate will result in a longer time lapse between synthesis of the upstream alternative exon and the downstream constitutive one than if transcribed by a faster polymerase. This lag will afford the spliceosome more time to recognize the weaker 3′ss of the alternative exon without competition from the downstream constitutive exon, resulting in increased alternative exon inclusion. Pausing of the polymerase within the intron downstream of the alternative exon could similarly permit increased splicing of the regulated exon. (c) In the case of FN alternative exon 33, the downstream intron is excised prior to the upstream one. In this case, it is thought that the rate of transcription alters commitment complex assembly on the pre‐mRNA rather than affecting the order of intron excision per se. In this case, splicing choices would be decided prior to intron excision.

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CTD phosphorylation recruits mRNA capping enzymes. The cdk7 subunit of TFIIH phosphorylates Ser5 residues on the CTD of promoter proximal RNA polII molecules. The pre‐mRNA capping enzymes bind to the CTD in a phospho‐Ser5 dependent manner and catalyze cap addition on the 5′ end of the pre‐mRNA. Ser5 residues are dephosphorylated sometime after transcriptional initiation, probably by the Rtr1 or SSu72 phosphatase(s). Phospho‐Ser2 marks are detected on elongating polymerases located within genes. The cdk9 subunit of P‐TEFb is the Ser2 kinase. P‐TEFb binds to promoter sequences and stimulates transcriptional elongation, but it is not known whether it has to be promoter bound to phosphorylate the CTD. RNA polymerase II can interact with RS domain‐containing proteins through recruitment mechanism(s) that are unclear, as indicated by a question mark.

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The spliceosome assembles in an ordered, step‐wise manner in vitro. In the E (early or commitment) complex the U1 snRNP binds the 5′ss via base pairing interactions, U2AF recognizes the PPT and additional factors bring the 5′ss and 3′ss into juxtaposition. This pairing is stabilized by addition of ATP, which allows binding of the U2 snRNP to the BP to form the A complex. The U4/U6–U5 tri‐snRNP then joins as a single unit to form the B complex. Multiple ATP‐dependent rearrangements result in the release of the U1 and U4 snRNPs and formation of the C complex, which catalyzes excision of the intron as a lariat and ligation of the exon sequences.

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Factors involved in 3′ end processing interact with members of the splicing apparatus. Double arrowheads indicate known interactions (direct and indirect) between splicing factors and the cleavage and polyadenylation machinery. For clarity, interactions that occur between spliceosomal factors during spliceosomal assembly, or between the cleavage and polyadenylation factors during 3′ end processing have been omitted. The annotation(s) next to each arrowhead indicate the citation number for the studies that describe the indicated interactions.

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Browse by Topic

RNA Processing > 3′ End Processing
RNA Processing > Capping and 5′ End Modifications
RNA Processing > Splicing Regulation/Alternative Splicing
RNA Processing > Splicing Mechanisms

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