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Regulation of mRNA abundance by polypyrimidine tract-binding protein-controlled alternate 5' splice site choice.

Hamid FM, Makeyev EV - PLoS Genet. (2014)

Bottom Line: Consistent with this mechanism, u5'ss is intrinsically weaker than d5'ss, with a similar tendency observed for other genes with Ptbp1-induced u5'ss bias.Interestingly, the brain-enriched Ptbp1 paralog Ptbp2/nPTB/brPTB stimulated the u5'ss utilization but with a considerably lower efficiency than Ptbp1.More generally, these data expand our understanding of AS regulation and uncover a post-transcriptional strategy ensuring co-expression of a subordinate gene with its master regulator through an AS-NMD tracking mechanism.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.

ABSTRACT
Alternative splicing (AS) provides a potent mechanism for increasing protein diversity and modulating gene expression levels. How alternate splice sites are selected by the splicing machinery and how AS is integrated into gene regulation networks remain important questions of eukaryotic biology. Here we report that polypyrimidine tract-binding protein 1 (Ptbp1/PTB/hnRNP-I) controls alternate 5' and 3' splice site (5'ss and 3'ss) usage in a large set of mammalian transcripts. A top scoring event identified by our analysis was the choice between competing upstream and downstream 5'ss (u5'ss and d5'ss) in the exon 18 of the Hps1 gene. Hps1 is essential for proper biogenesis of lysosome-related organelles and loss of its function leads to a disease called type 1 Hermansky-Pudlak Syndrome (HPS). We show that Ptbp1 promotes preferential utilization of the u5'ss giving rise to stable mRNAs encoding a full-length Hps1 protein, whereas bias towards d5'ss triggered by Ptbp1 down-regulation generates transcripts susceptible to nonsense-mediated decay (NMD). We further demonstrate that Ptbp1 binds to pyrimidine-rich sequences between the u5'ss and d5'ss and activates the former site rather than repressing the latter. Consistent with this mechanism, u5'ss is intrinsically weaker than d5'ss, with a similar tendency observed for other genes with Ptbp1-induced u5'ss bias. Interestingly, the brain-enriched Ptbp1 paralog Ptbp2/nPTB/brPTB stimulated the u5'ss utilization but with a considerably lower efficiency than Ptbp1. This may account for the tight correlation between Hps1 with Ptbp1 expression levels observed across mammalian tissues. More generally, these data expand our understanding of AS regulation and uncover a post-transcriptional strategy ensuring co-expression of a subordinate gene with its master regulator through an AS-NMD tracking mechanism.

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Hps1 regulation depends on u5′ss being weaker than d5′ss.(A) TRE-mini-1819 minigenes containing either a wild-type (top) or a permuted arrangement of the two 5′ss. (B) CAD cells pre-treated with indicated siRNAs were transfected with the TRE-mini-1819 constructs introduced in (A) and analyzed by RT-PCR using minigene-specific primers F1/R4. Note that the upstream 5′ splice position is constitutively used in all permuted minigene samples. (C) Utilization of the topologically downstream 5′ splice site [ψ(down)] in (B) averaged from three independent experiments ±SD.
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pgen-1004771-g006: Hps1 regulation depends on u5′ss being weaker than d5′ss.(A) TRE-mini-1819 minigenes containing either a wild-type (top) or a permuted arrangement of the two 5′ss. (B) CAD cells pre-treated with indicated siRNAs were transfected with the TRE-mini-1819 constructs introduced in (A) and analyzed by RT-PCR using minigene-specific primers F1/R4. Note that the upstream 5′ splice position is constitutively used in all permuted minigene samples. (C) Utilization of the topologically downstream 5′ splice site [ψ(down)] in (B) averaged from three independent experiments ±SD.

Mentions: Our data so far suggested that Ptbp1 interacts with Py1 and Py2 sequences within exon 18 and enhances u5′ss utilization. Interestingly, predicted splicing strength of u5′ss was lower than that of d5′ss (scores Su5′ss = 76.1 vs. Sd5′ss = 94.1 obtained using Analyzer Splice Tool server http://ibis.tau.ac.il/ssat/SpliceSiteFrame.htm; [51], [52]) and similar differences were detected in other mammalian species (Table S5). To test if this feature was important for the regulation, we generated a series of modified TRE-mini-1819 minigenes where the natural u5′ss was substituted with the d5′ss or/and the d5′ss was substituted with the u5′ss [TRE-mini-1819(d5′ss/d5′ss), TRE-mini-1819(u5′ss/u5′ss) and TRE-mini-1819(d5′ss/u5′ss); Fig. 6A]. All of these permutations lowered the ΔS = Sd5′ss-Su5′ss difference between the two 5′ss strengths. Notably, when we transfected CAD cells with the corresponding minigenes, the upstream 5′ splice position was constitutively selected in all siRNA-treated samples (Fig. 6B–C). Similar effects were observed when we weakened the u5′ss or strengthened the d5′ss by substituting them with synthetic 5′ss sequences (Fig. S9). These results are consistent with the model that Hps1 A5C regulation requires u5′ss to be weaker than d5′ss.


Regulation of mRNA abundance by polypyrimidine tract-binding protein-controlled alternate 5' splice site choice.

Hamid FM, Makeyev EV - PLoS Genet. (2014)

Hps1 regulation depends on u5′ss being weaker than d5′ss.(A) TRE-mini-1819 minigenes containing either a wild-type (top) or a permuted arrangement of the two 5′ss. (B) CAD cells pre-treated with indicated siRNAs were transfected with the TRE-mini-1819 constructs introduced in (A) and analyzed by RT-PCR using minigene-specific primers F1/R4. Note that the upstream 5′ splice position is constitutively used in all permuted minigene samples. (C) Utilization of the topologically downstream 5′ splice site [ψ(down)] in (B) averaged from three independent experiments ±SD.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222953&req=5

pgen-1004771-g006: Hps1 regulation depends on u5′ss being weaker than d5′ss.(A) TRE-mini-1819 minigenes containing either a wild-type (top) or a permuted arrangement of the two 5′ss. (B) CAD cells pre-treated with indicated siRNAs were transfected with the TRE-mini-1819 constructs introduced in (A) and analyzed by RT-PCR using minigene-specific primers F1/R4. Note that the upstream 5′ splice position is constitutively used in all permuted minigene samples. (C) Utilization of the topologically downstream 5′ splice site [ψ(down)] in (B) averaged from three independent experiments ±SD.
Mentions: Our data so far suggested that Ptbp1 interacts with Py1 and Py2 sequences within exon 18 and enhances u5′ss utilization. Interestingly, predicted splicing strength of u5′ss was lower than that of d5′ss (scores Su5′ss = 76.1 vs. Sd5′ss = 94.1 obtained using Analyzer Splice Tool server http://ibis.tau.ac.il/ssat/SpliceSiteFrame.htm; [51], [52]) and similar differences were detected in other mammalian species (Table S5). To test if this feature was important for the regulation, we generated a series of modified TRE-mini-1819 minigenes where the natural u5′ss was substituted with the d5′ss or/and the d5′ss was substituted with the u5′ss [TRE-mini-1819(d5′ss/d5′ss), TRE-mini-1819(u5′ss/u5′ss) and TRE-mini-1819(d5′ss/u5′ss); Fig. 6A]. All of these permutations lowered the ΔS = Sd5′ss-Su5′ss difference between the two 5′ss strengths. Notably, when we transfected CAD cells with the corresponding minigenes, the upstream 5′ splice position was constitutively selected in all siRNA-treated samples (Fig. 6B–C). Similar effects were observed when we weakened the u5′ss or strengthened the d5′ss by substituting them with synthetic 5′ss sequences (Fig. S9). These results are consistent with the model that Hps1 A5C regulation requires u5′ss to be weaker than d5′ss.

Bottom Line: Consistent with this mechanism, u5'ss is intrinsically weaker than d5'ss, with a similar tendency observed for other genes with Ptbp1-induced u5'ss bias.Interestingly, the brain-enriched Ptbp1 paralog Ptbp2/nPTB/brPTB stimulated the u5'ss utilization but with a considerably lower efficiency than Ptbp1.More generally, these data expand our understanding of AS regulation and uncover a post-transcriptional strategy ensuring co-expression of a subordinate gene with its master regulator through an AS-NMD tracking mechanism.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.

ABSTRACT
Alternative splicing (AS) provides a potent mechanism for increasing protein diversity and modulating gene expression levels. How alternate splice sites are selected by the splicing machinery and how AS is integrated into gene regulation networks remain important questions of eukaryotic biology. Here we report that polypyrimidine tract-binding protein 1 (Ptbp1/PTB/hnRNP-I) controls alternate 5' and 3' splice site (5'ss and 3'ss) usage in a large set of mammalian transcripts. A top scoring event identified by our analysis was the choice between competing upstream and downstream 5'ss (u5'ss and d5'ss) in the exon 18 of the Hps1 gene. Hps1 is essential for proper biogenesis of lysosome-related organelles and loss of its function leads to a disease called type 1 Hermansky-Pudlak Syndrome (HPS). We show that Ptbp1 promotes preferential utilization of the u5'ss giving rise to stable mRNAs encoding a full-length Hps1 protein, whereas bias towards d5'ss triggered by Ptbp1 down-regulation generates transcripts susceptible to nonsense-mediated decay (NMD). We further demonstrate that Ptbp1 binds to pyrimidine-rich sequences between the u5'ss and d5'ss and activates the former site rather than repressing the latter. Consistent with this mechanism, u5'ss is intrinsically weaker than d5'ss, with a similar tendency observed for other genes with Ptbp1-induced u5'ss bias. Interestingly, the brain-enriched Ptbp1 paralog Ptbp2/nPTB/brPTB stimulated the u5'ss utilization but with a considerably lower efficiency than Ptbp1. This may account for the tight correlation between Hps1 with Ptbp1 expression levels observed across mammalian tissues. More generally, these data expand our understanding of AS regulation and uncover a post-transcriptional strategy ensuring co-expression of a subordinate gene with its master regulator through an AS-NMD tracking mechanism.

Show MeSH
Related in: MedlinePlus