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iCLIP predicts the dual splicing effects of TIA-RNA interactions.

Wang Z, Kayikci M, Briese M, Zarnack K, Luscombe NM, Rot G, Zupan B, Curk T, Ule J - PLoS Biol. (2010)

Bottom Line: However, effects of TIA proteins on splicing of distal exons have not yet been explored.Binding downstream of 5' splice sites was used to predict the effects of TIA proteins in enhancing inclusion of proximal exons and silencing inclusion of distal exons.Thus, our findings indicate that changes in splicing kinetics could mediate the distal regulation of alternative splicing.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council - Laboratory of Molecular Biology, Hills Road, Cambridge, United Kingdom.

ABSTRACT
The regulation of alternative splicing involves interactions between RNA-binding proteins and pre-mRNA positions close to the splice sites. T-cell intracellular antigen 1 (TIA1) and TIA1-like 1 (TIAL1) locally enhance exon inclusion by recruiting U1 snRNP to 5' splice sites. However, effects of TIA proteins on splicing of distal exons have not yet been explored. We used UV-crosslinking and immunoprecipitation (iCLIP) to find that TIA1 and TIAL1 bind at the same positions on human RNAs. Binding downstream of 5' splice sites was used to predict the effects of TIA proteins in enhancing inclusion of proximal exons and silencing inclusion of distal exons. The predictions were validated in an unbiased manner using splice-junction microarrays, RT-PCR, and minigene constructs, which showed that TIA proteins maintain splicing fidelity and regulate alternative splicing by binding exclusively downstream of 5' splice sites. Surprisingly, TIA binding at 5' splice sites silenced distal cassette and variable-length exons without binding in proximity to the regulated alternative 3' splice sites. Using transcriptome-wide high-resolution mapping of TIA-RNA interactions we evaluated the distal splicing effects of TIA proteins. These data are consistent with a model where TIA proteins shorten the time available for definition of an alternative exon by enhancing recognition of the preceding 5' splice site. Thus, our findings indicate that changes in splicing kinetics could mediate the distal regulation of alternative splicing.

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An overview of the models of TIA-dependent splicing regulation.(A) TIA proteins can directly regulate 5′ splice site competition by enhancing either intron-proximal or intron-distal 5′ splice sites. (B) TIA proteins directly regulate alternative cassette exon inclusion by enhancing 5′ splice sites. (C) TIA proteins promote the use of intron-distal alternative 3′ splice sites without directly modulating competition between the alternative 3′ splice sites. By enhancing 5′ splice site recognition, TIA proteins decrease the inclusion of the variable portion of the exon. The splicing kinetics model proposes that the splicing kinetics is affected by 5′ splice site recognition, which then indirectly affects the ability of SR proteins and other factors to define the variable portion of the distal exon. According to this model, the slower splicing kinetics in the absence of TIA increases the time available to these factors to promote inclusion of the variable portion of the exon. (D) Similar to the effect on distal variable exons, a change in splicing kinetics could contribute to the ability of TIA proteins to promote skipping of distal alternative cassette exon by binding at the upstream 5′ splice site.
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pbio-1000530-g007: An overview of the models of TIA-dependent splicing regulation.(A) TIA proteins can directly regulate 5′ splice site competition by enhancing either intron-proximal or intron-distal 5′ splice sites. (B) TIA proteins directly regulate alternative cassette exon inclusion by enhancing 5′ splice sites. (C) TIA proteins promote the use of intron-distal alternative 3′ splice sites without directly modulating competition between the alternative 3′ splice sites. By enhancing 5′ splice site recognition, TIA proteins decrease the inclusion of the variable portion of the exon. The splicing kinetics model proposes that the splicing kinetics is affected by 5′ splice site recognition, which then indirectly affects the ability of SR proteins and other factors to define the variable portion of the distal exon. According to this model, the slower splicing kinetics in the absence of TIA increases the time available to these factors to promote inclusion of the variable portion of the exon. (D) Similar to the effect on distal variable exons, a change in splicing kinetics could contribute to the ability of TIA proteins to promote skipping of distal alternative cassette exon by binding at the upstream 5′ splice site.

Mentions: In the present study, we have evaluated both the local and distal splicing effects of TIA binding at 5′ splice sites. Locally, TIA proteins can regulate alternative 5′ splice sites in a manner consistent with the splice site competition model [41]. TIA proteins enhanced the closest upstream 5′ splice site, leading to a concomitant decrease in usage of the competing 5′ splice site (Figure 7A). Similarly, TIA binding downstream of a cassette exon acts by promoting the usage of its 5′ splice site (Figure 7B). A past study evaluating splicing intermediates showed that in cases of Nova binding downstream of enhanced exons, the downstream intron is removed prior to the upstream one [4]. This result is consistent with a splice site competition model, indicating that Nova and TIA proteins can enhance cassette exons by promoting the splicing pathway that uses the 5′ splice site of the alternative exon, with concomitant decrease in the exon skipping pathway that uses the 5′ splice site of the preceding exon (Figure 7B).


iCLIP predicts the dual splicing effects of TIA-RNA interactions.

Wang Z, Kayikci M, Briese M, Zarnack K, Luscombe NM, Rot G, Zupan B, Curk T, Ule J - PLoS Biol. (2010)

An overview of the models of TIA-dependent splicing regulation.(A) TIA proteins can directly regulate 5′ splice site competition by enhancing either intron-proximal or intron-distal 5′ splice sites. (B) TIA proteins directly regulate alternative cassette exon inclusion by enhancing 5′ splice sites. (C) TIA proteins promote the use of intron-distal alternative 3′ splice sites without directly modulating competition between the alternative 3′ splice sites. By enhancing 5′ splice site recognition, TIA proteins decrease the inclusion of the variable portion of the exon. The splicing kinetics model proposes that the splicing kinetics is affected by 5′ splice site recognition, which then indirectly affects the ability of SR proteins and other factors to define the variable portion of the distal exon. According to this model, the slower splicing kinetics in the absence of TIA increases the time available to these factors to promote inclusion of the variable portion of the exon. (D) Similar to the effect on distal variable exons, a change in splicing kinetics could contribute to the ability of TIA proteins to promote skipping of distal alternative cassette exon by binding at the upstream 5′ splice site.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2964331&req=5

pbio-1000530-g007: An overview of the models of TIA-dependent splicing regulation.(A) TIA proteins can directly regulate 5′ splice site competition by enhancing either intron-proximal or intron-distal 5′ splice sites. (B) TIA proteins directly regulate alternative cassette exon inclusion by enhancing 5′ splice sites. (C) TIA proteins promote the use of intron-distal alternative 3′ splice sites without directly modulating competition between the alternative 3′ splice sites. By enhancing 5′ splice site recognition, TIA proteins decrease the inclusion of the variable portion of the exon. The splicing kinetics model proposes that the splicing kinetics is affected by 5′ splice site recognition, which then indirectly affects the ability of SR proteins and other factors to define the variable portion of the distal exon. According to this model, the slower splicing kinetics in the absence of TIA increases the time available to these factors to promote inclusion of the variable portion of the exon. (D) Similar to the effect on distal variable exons, a change in splicing kinetics could contribute to the ability of TIA proteins to promote skipping of distal alternative cassette exon by binding at the upstream 5′ splice site.
Mentions: In the present study, we have evaluated both the local and distal splicing effects of TIA binding at 5′ splice sites. Locally, TIA proteins can regulate alternative 5′ splice sites in a manner consistent with the splice site competition model [41]. TIA proteins enhanced the closest upstream 5′ splice site, leading to a concomitant decrease in usage of the competing 5′ splice site (Figure 7A). Similarly, TIA binding downstream of a cassette exon acts by promoting the usage of its 5′ splice site (Figure 7B). A past study evaluating splicing intermediates showed that in cases of Nova binding downstream of enhanced exons, the downstream intron is removed prior to the upstream one [4]. This result is consistent with a splice site competition model, indicating that Nova and TIA proteins can enhance cassette exons by promoting the splicing pathway that uses the 5′ splice site of the alternative exon, with concomitant decrease in the exon skipping pathway that uses the 5′ splice site of the preceding exon (Figure 7B).

Bottom Line: However, effects of TIA proteins on splicing of distal exons have not yet been explored.Binding downstream of 5' splice sites was used to predict the effects of TIA proteins in enhancing inclusion of proximal exons and silencing inclusion of distal exons.Thus, our findings indicate that changes in splicing kinetics could mediate the distal regulation of alternative splicing.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council - Laboratory of Molecular Biology, Hills Road, Cambridge, United Kingdom.

ABSTRACT
The regulation of alternative splicing involves interactions between RNA-binding proteins and pre-mRNA positions close to the splice sites. T-cell intracellular antigen 1 (TIA1) and TIA1-like 1 (TIAL1) locally enhance exon inclusion by recruiting U1 snRNP to 5' splice sites. However, effects of TIA proteins on splicing of distal exons have not yet been explored. We used UV-crosslinking and immunoprecipitation (iCLIP) to find that TIA1 and TIAL1 bind at the same positions on human RNAs. Binding downstream of 5' splice sites was used to predict the effects of TIA proteins in enhancing inclusion of proximal exons and silencing inclusion of distal exons. The predictions were validated in an unbiased manner using splice-junction microarrays, RT-PCR, and minigene constructs, which showed that TIA proteins maintain splicing fidelity and regulate alternative splicing by binding exclusively downstream of 5' splice sites. Surprisingly, TIA binding at 5' splice sites silenced distal cassette and variable-length exons without binding in proximity to the regulated alternative 3' splice sites. Using transcriptome-wide high-resolution mapping of TIA-RNA interactions we evaluated the distal splicing effects of TIA proteins. These data are consistent with a model where TIA proteins shorten the time available for definition of an alternative exon by enhancing recognition of the preceding 5' splice site. Thus, our findings indicate that changes in splicing kinetics could mediate the distal regulation of alternative splicing.

Show MeSH
Related in: MedlinePlus