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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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TIA1 and TIAL1 crosslink to the same positions in human RNAs.(A) The percentage of cDNAs from TIA1 and TIAL1 iCLIP that mapped to different types of RNAs. (B) The fold enrichment of average cDNA density from TIA1 and TIAL1 iCLIP in different types of RNAs relative to the average cDNA density in the whole genome. (C) Pentamer z scores at the 21 nt sequence surrounding crosslink sites (−10 nt to +10 nt) are shown for TIA1 and TIAL1 iCLIP. The sequences of the two most enriched pentamers and the Pearson correlation coefficient (r) between the TIA1 and TIAL1 z scores are shown. (D) Reproducibility of TIA1 and TIAL1 crosslink clusters. Percentage of crosslink clusters with a given cDNA count in TIA1 iCLIP that were also identified in TIAL1 iCLIP is shown. (E) Contour plot comparing TIA1 and TIAL1 cDNA counts in the 46,970 crosslink clusters. The darkness of contours increases with the number of clusters. The Pearson correlation coefficient (r) between the TIA1 and TIAL1 cDNA counts in the clusters is shown. (F) Crosslink sites of TIA1 and TIAL1 in the 3′ UTR of the MYC gene. The bar graph shows the number of cDNAs that identified each crosslink site. The Pearson correlation coefficient (r) between the TIA1 and TIAL1 cDNA counts at individual nucleotides is shown.
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pbio-1000530-g002: TIA1 and TIAL1 crosslink to the same positions in human RNAs.(A) The percentage of cDNAs from TIA1 and TIAL1 iCLIP that mapped to different types of RNAs. (B) The fold enrichment of average cDNA density from TIA1 and TIAL1 iCLIP in different types of RNAs relative to the average cDNA density in the whole genome. (C) Pentamer z scores at the 21 nt sequence surrounding crosslink sites (−10 nt to +10 nt) are shown for TIA1 and TIAL1 iCLIP. The sequences of the two most enriched pentamers and the Pearson correlation coefficient (r) between the TIA1 and TIAL1 z scores are shown. (D) Reproducibility of TIA1 and TIAL1 crosslink clusters. Percentage of crosslink clusters with a given cDNA count in TIA1 iCLIP that were also identified in TIAL1 iCLIP is shown. (E) Contour plot comparing TIA1 and TIAL1 cDNA counts in the 46,970 crosslink clusters. The darkness of contours increases with the number of clusters. The Pearson correlation coefficient (r) between the TIA1 and TIAL1 cDNA counts in the clusters is shown. (F) Crosslink sites of TIA1 and TIAL1 in the 3′ UTR of the MYC gene. The bar graph shows the number of cDNAs that identified each crosslink site. The Pearson correlation coefficient (r) between the TIA1 and TIAL1 cDNA counts at individual nucleotides is shown.

Mentions: The random barcode introduced into iCLIP cDNAs allowed us to analyse the distribution of TIA1 and TIAL1 on human RNAs in a quantitative and reproducible manner (Figure S2). Only 1.7% of cDNAs mapped in antisense orientation to annotated genes, confirming the high strand specificity of iCLIP. Only 10% of cDNAs mapped to intergenic regions (Figure 2A). The highest cDNA density was seen in 3′ UTRs and ncRNAs, which together contained 22% of all cDNAs (Figure 2A and 2B). 2,277 ncRNAs and 8,602 3′ UTRs had a higher cDNA density than the whole-genome average, and the cDNA enrichment correlated between TIA1 and TIAL1 iCLIP (Pearson correlation coefficient r = 0.95 and r = 0.90, respectively; Figure S3G and S3H). The ncRNA and 3′ UTR sites with the highest cDNA counts mapped to highly expressed RNAs such as tRNAs and histone mRNAs (Figure S4B). Interestingly, cDNA enrichment in 3′ UTRs was 5-fold higher than in the coding sequence (Figure 2B), in agreement with past findings that TIA proteins bind 3′ UTR to regulate translation [18],[29]–[31].


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)

TIA1 and TIAL1 crosslink to the same positions in human RNAs.(A) The percentage of cDNAs from TIA1 and TIAL1 iCLIP that mapped to different types of RNAs. (B) The fold enrichment of average cDNA density from TIA1 and TIAL1 iCLIP in different types of RNAs relative to the average cDNA density in the whole genome. (C) Pentamer z scores at the 21 nt sequence surrounding crosslink sites (−10 nt to +10 nt) are shown for TIA1 and TIAL1 iCLIP. The sequences of the two most enriched pentamers and the Pearson correlation coefficient (r) between the TIA1 and TIAL1 z scores are shown. (D) Reproducibility of TIA1 and TIAL1 crosslink clusters. Percentage of crosslink clusters with a given cDNA count in TIA1 iCLIP that were also identified in TIAL1 iCLIP is shown. (E) Contour plot comparing TIA1 and TIAL1 cDNA counts in the 46,970 crosslink clusters. The darkness of contours increases with the number of clusters. The Pearson correlation coefficient (r) between the TIA1 and TIAL1 cDNA counts in the clusters is shown. (F) Crosslink sites of TIA1 and TIAL1 in the 3′ UTR of the MYC gene. The bar graph shows the number of cDNAs that identified each crosslink site. The Pearson correlation coefficient (r) between the TIA1 and TIAL1 cDNA counts at individual nucleotides is shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2964331&req=5

pbio-1000530-g002: TIA1 and TIAL1 crosslink to the same positions in human RNAs.(A) The percentage of cDNAs from TIA1 and TIAL1 iCLIP that mapped to different types of RNAs. (B) The fold enrichment of average cDNA density from TIA1 and TIAL1 iCLIP in different types of RNAs relative to the average cDNA density in the whole genome. (C) Pentamer z scores at the 21 nt sequence surrounding crosslink sites (−10 nt to +10 nt) are shown for TIA1 and TIAL1 iCLIP. The sequences of the two most enriched pentamers and the Pearson correlation coefficient (r) between the TIA1 and TIAL1 z scores are shown. (D) Reproducibility of TIA1 and TIAL1 crosslink clusters. Percentage of crosslink clusters with a given cDNA count in TIA1 iCLIP that were also identified in TIAL1 iCLIP is shown. (E) Contour plot comparing TIA1 and TIAL1 cDNA counts in the 46,970 crosslink clusters. The darkness of contours increases with the number of clusters. The Pearson correlation coefficient (r) between the TIA1 and TIAL1 cDNA counts in the clusters is shown. (F) Crosslink sites of TIA1 and TIAL1 in the 3′ UTR of the MYC gene. The bar graph shows the number of cDNAs that identified each crosslink site. The Pearson correlation coefficient (r) between the TIA1 and TIAL1 cDNA counts at individual nucleotides is shown.
Mentions: The random barcode introduced into iCLIP cDNAs allowed us to analyse the distribution of TIA1 and TIAL1 on human RNAs in a quantitative and reproducible manner (Figure S2). Only 1.7% of cDNAs mapped in antisense orientation to annotated genes, confirming the high strand specificity of iCLIP. Only 10% of cDNAs mapped to intergenic regions (Figure 2A). The highest cDNA density was seen in 3′ UTRs and ncRNAs, which together contained 22% of all cDNAs (Figure 2A and 2B). 2,277 ncRNAs and 8,602 3′ UTRs had a higher cDNA density than the whole-genome average, and the cDNA enrichment correlated between TIA1 and TIAL1 iCLIP (Pearson correlation coefficient r = 0.95 and r = 0.90, respectively; Figure S3G and S3H). The ncRNA and 3′ UTR sites with the highest cDNA counts mapped to highly expressed RNAs such as tRNAs and histone mRNAs (Figure S4B). Interestingly, cDNA enrichment in 3′ UTRs was 5-fold higher than in the coding sequence (Figure 2B), in agreement with past findings that TIA proteins bind 3′ UTR to regulate translation [18],[29]–[31].

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