Mechanism underlying PTBP1-mediated alternative pre-mRNA splicing in regulating tissue-specific gene expression

• Main Author: Muhammad Fursham Hamid
• Other Authors: Eugene Makeyev
• Format: Theses and Dissertations
• Language: English
• Published: 2016
• Subjects: DRNTU::Science::Biological sciences::Molecular biology
• Online Access: https://hdl.handle.net/10356/69374
• Institution: Nanyang Technological University

Alternative pre-mRNA splicing (AS) provides an efficient means for diversifying the proteome and regulating gene expression levels in the higher eukaryotes. The gene regulation function often involves coupling between AS and nonsense-mediated decay (NMD), a cytoplasmic surveillance mechanism destabilizing mRNAs with premature termination codons. AS is controlled by interplay between cis-regulatory elements and trans-acting factors modulating the usage of alternative pre-mRNA segments. Polypyrimidine tract-binding protein 1 (Ptbp1/PTB/ hnRNP-I), a global repressor of neuron-specific alternative splicing program in non-neuronal cells, has been previously shown to repress several neuronal genes by inhibiting the inclusion of open reading frame-maintaining exons and subjecting the exon-skipped transcripts to NMD. Down-regulation of Ptbp1 levels in developing neurons alleviates this effect and gives rise to translation-competent transcripts. Here we identified several new Ptbp1-dependent AS-NMD targets that, unlike the earlier described examples, require Ptbp1 for their optimal expression. One such target, the Hermansky-Pudlak Syndrome 1 (Hps1) gene, is regulated by a unique Ptbp1-mediated choice between two competing alternative 5’ splice sites (5’ss). Dampening Ptbp1 levels promotes the usage of the downstream 5’ss and gives rise to an NMD-susceptible Hps1 mRNA isoform in neurons. We show that Ptbp1 functions in this circuitry by activating the upstream 5’ss rather than repressing the downstream 5’ss. This was surprising since Ptbp1 typically functions as a splicing repressor. To understand molecular mechanisms underlying Ptbp1-mediated splicing activation, we examined both Hps1 and another gene, Deltex2, containing a Ptbp1-dependent cassette exon. In both systems, Ptbp1 promotes the recruitment of a core spliceosomal component, U1 snRNP, to a weak 5’ss by interacting with a specific structural element in the U1 snRNA.