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Exon and intron definition in pre‐mRNA splicing

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Abstract One of the fundamental issues in RNA splicing research is represented by understanding how the spliceosome can successfully define exons and introns in a huge variety of pre‐mRNA molecules with nucleotide‐precision. Since its first description, researchers in this field have identified and characterized many fundamental elements and players capable of affecting the splicing process, both in a negative and positive manner. Indeed, it can be argued that today we know a great deal about the forces that make an exon, an exon and an intron, an intron. As will be discussed in this review, these decisions are a result of a complex combinatorial control resulting from many different factors/influences. Most importantly, these influences act across several levels of complexity starting from the relatively simple interaction between two consensus 5′ and 3′ splice sites to much more complex factors: such as the interplay between silencer or enhancer sequences, transcriptional processivity, genomic milieu, nucleosome positioning, and histone modifications at the chromatin level. Depending on local contexts, all these factors will act either antagonistically or synergistically to decide the exon/intron fate of any given RNA sequence. At present, however, what we still lack is a precise understanding of how all these processes add up to help the spliceosome reach a decision. Therefore, it is expected that future challenges in splicing research will be the careful characterization of all these influences to improve our ability to predict splicing choices in different organisms or in specific contexts. WIREs RNA 2013, 4:49–60. doi: 10.1002/wrna.1140 This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing

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The splicing process. (a) Gray boxes represent exons, separated by intervening sequences (introns) shown as lines. Conserved, canonical splice signals (GU, AG) are indicated at the 3′ and 5′ ends of the exons (R means A/G). Other 3′ splice site signatures are represented by the branch site (BS) and the polypyrimidine tract (Py). (b) snRNP U1 bound to the 5′ ss and U2AF35 together with U2AF65 bound to the AG dinucleotide and the polypyrimidine tract. The branch site is subsequently bound by SF1/mBBP. These splicing factors form the E complex (boxed) that bridges the intron and brings the splice sites together. Subsequently (c), the U2 snRNP displaces SF1/mBBP from the BS giving rise to the A complex (boxed). The next step requires the binding of U4/U5·U6 tri‐snRNP and the forming the precatalytic B complex (boxed) (d). The next step sees the destabilization of U1 and U4 snRNPs and, together with the association of the Prp19/CDC5L complex, represents one of the key events in the activation of the B complex (B* complex). (e) The activated B complex catalyzes the first of the two steps of the splicing. Finally, the catalytically competent complex C is formed. To note that exonic splicing enhancers and silencers can sinergistically or antagonistically influence the spliceosome formation efficiency. In this figure they are represented by light grey boxes and their binding factors are red or blue circles according to their positive or negative contribution. (f) Schematic representation of the recognition of the terminal exons. Definition of the 5′‐terminal exon requires the cap‐binding protein complex to communicate with the 5′ss. On the other hand, definition of the 3′‐terminal exon needs the communication between the 3′ss and the poly(A) factors (among which the cleavage/polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factor (CF), and poly(A) polymerase (PAP)).

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Model for KCNH2 exon 7 ESE in stabilizing an exon defined spliceosome. Schematic representation of the KCNH2 exon 7 minigene construct: the black box represents the KCNH2 exon 7 with intervening sequences shown as thin lines. Grey boxes represent the flanking minigene exons. The intronic mutation +6T>C is shown as a black dot. The purine rich ESE is shown in its wild type form (panel a) and in its neutralized form (panel b). (a) In presence of +6 T>C the U1snRNP binding to the 5′ss is inhibited (depicted as a dashed snRNP U1). Nevertheless, a weak cross‐exon communication, enhanced by the ESE (red arrow), between the 5′ and 3′ss still occurs. Because the 3′ss of KCNH2 exon 7 is still recognized by the splicing machinery this results in intron retention. (b) Once the ESE is neutralized by mutations (KCNH2 +6T>C PurMut) the cross‐exon communication is repressed (blue line) and as a consequence the 3′ss is no longer recognized (dashed U2AF35 and U2AF65). This condition results predominantly in the skipping of the exon. Solid arrows represent functional communications between the various sites while dashed arrows indicate absent or incomplete communication.

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Model for PTPRC/CD45 exon 4 silencer in repressing proper exon splicing during the intron definition transition. (a) In absence of silencer binding factors exon 4 is included in the final transcript. The light grey box in exon 4 represents the ESS. (b) In presence of the PTPRC/CD45 exon 4 silencer binding factors (among which hnRNP L here represented as a blue circle) it has been hypothesized that a strong ternary complex with snRNP U1 and U2 is formed. This interaction is more favourable than the one with the snRNPs bound to the flanking exons resulting in the exclusion of exon 4 from the mature transcript.

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Exon and Intron definition models. The left panel depicts the Intron definition model according to which pairing between the splice sites takes place across an intron when long exons are separated by short (<250 bp) introns. On the other hand the right panel shows the Exon definition model where the splice site communication occurs across exons when they are separated by long (>250 bp) introns.

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RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition
RNA Processing > Splicing Regulation/Alternative Splicing
RNA Processing > Splicing Mechanisms

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