Date of Award
Doctor of Philosophy
Ravindra N. Singh
Humans carry two near identical copies of Survival Motor Neuron gene: SMN1 and SMN2. Deletion or a mutation(s) of SMN1 coupled with the predominant skipping of SMN2 exon 7 causes SMA, the most frequent genetic cause of infant mortality. Correction of SMN2 exon 7 splicing holds the promise for cure. Hence it is crucial to understand the regulation of SMN2 splicing and function of SMN isoforms. We employ a multiple-exon-skipping detection assay (MESDA) to capture the relative abundance of all major transcripts in one reaction. Our results show surprising diversity of splice isoforms and reveal novel splicing events that include skipping of exon 4, co-skipping of three adjacent exons of SMN and exonization of a portion of intron 6. In cell-based studies, our results reveal oxidative stress (OS) induced skipping of SMN1 exon 5 in several cell types, including non-neuronal cells. In in vivo study, we observed that unique tissue-specific signature of SMN2 splice isoforms generated under OS. We also show profound differences between male and female reproductive organs with respect to the SMN2 splice isoforms produced under normal and OS conditions. Employing a super minigene harboring SMN2 promoter, all internal exons and flanking intronic sequences, we demonstrate transcription-coupled splicing regulation of various SMN2 exons under normal and OS conditions. Our results of proteome analysis of mouse brain revealed OS-induced downregulation of hnRNP H, a multifunctional proteins, involved in pre-mRNA splicing and 3′-end processing. We confirm hnRNP H as a positive regulator of SMN levels and likely serves as a disease-modifying factor for SMA. Taken together, a number of important observations expand the diversity of splice isoforms generated by SMN and bring new insight into our understanding of splicing regulation of SMN.
Seo, Joonbae, "Characterization of splice variants of human Survival Motor Neuron genes" (2015). Graduate Theses and Dissertations. 14741.