Alternative pre-mRNA splicing in mammalian nervous system

• Main Author: Yap, Karen Lin Choo
• Other Authors: School of Biological Sciences
• Format: Theses and Dissertations
• Language: English
• Published: 2014
• Subjects: DRNTU::Science
• Online Access: http://hdl.handle.net/10356/55438
• Institution: Nanyang Technological University

Alternative pre-mRNA splicing provides a potent post-transcriptional mechanism for diversifying the transcriptome and regulating gene expression levels. Polypyrimidine tract-binding protein (Ptbp1) is a global regulator of alternative splicing affecting approximately a quater of the neuron-specific splicing events. Ptbp1 is expressed at high level in multiple cell types but down-regulated in neurons by brain-enriched microRNA miR-124. Many previous studies have examined Ptbp1-mediated regulation of cassette exons, internal exons that can be alternatively included or skipped. Here I describe two additional Ptbp1 functions. First, I show that Ptbp1 regulates utilization of alternate 3’-terminal exons in a subset of genes operating in cell projection pathways. One such gene, Cdc42, contains two alternate 3’-terminal exons. Of these, exon 7 is utilized in a ubiquitous manner, whereas exon 6 is used exclusively in neurons since it is repressed by Ptbp1 in other cell types. I demonstrate that Ptbp1 inhibits exon 6 and biases splicing pattern towards exon 7 inclusion by interacting with intronic and exonic splicing silencer elements flanking the exon 6-specific 3’ splice site. I further address the biological function of this splicing switch by generating Cdc42 exon 6-null mice that constitutively produce exon 7-included mRNAs in all cell types. Detailed analyses of the mutant neurons show that the exon 6-included Cdc42 splice form is essential to maintain appropriate dendritic spine densities and, surprisingly, to limit the number of axons to one per neuron. In the second part of the thesis, I show that Ptbp1 represses splicing of 3’-terminal introns in pre-mRNAs encoding several pre-synaptic proteins thus enabling post-transcriptional regulation of mRNA steady state levels. In non-neuronal cells expressing large amounts of Ptbp1 protein, intron-containing transcrips are sequestered in the nucleus and eventually degraded mediated by nucleoporin Tpr and the nuclear exosome complex. However, a decrease in Ptbp1 expression in neurons allows the 3’-terminal introns to be spliced out, which promotes nucleocytoplasmic export and translation of the completely spliced mRNAs and ultimately, streamlines neuronal differentiation. Overall, these data expand our understanding of post-transcriptional regulation mechanisms operating in the context of developing nervous system.