Characterization of circular RNAs of Survival Motor Neuron genes
Date
2022-12
Authors
Luo, Diou
Major Professor
Advisor
Singh, Ravindra N
Singh, Natalia N
Cho, Michael
Kim, Jinoh
Miller, Cathy
Committee Member
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Abstract
Spinal Muscular Atrophy (SMA) is a destructive genetic disease associated with infant and child mortality with a carrier frequency of ~1 in 51 and affecting ~1 in 10,000 live births. Deficiency of the Survival Motor Neuron (SMN) protein owing to homologous disruption or deletion of the SMN1 gene accounts for more than ~90% of SMA cases. So far, most functional studies of the SMN genes are channeled to the SMN protein and its isoforms. However, limited information has been explored regarding SMN transcripts. This study first identified a broad spectrum of circular RNAs (circRNAs) generated by SMN genes involving all the annotated exons by using a tailored approach highlighted with RNase R treatment and PCR with divergent primers. We confirmed our findings in both cells and/or tissues from humans and mice. The result implied that DHX9, an RNA helicase, plays a role in regulating several SMN circRNAs. This study also showed the cross-regulation of SMN circRNA incorporated early exons. And still, the mechanism of SMN circRNA biogenesis remains largely unexplored. Next, constructs and stable cell lines were generated to overexpress three of the most abundantly expressed SMN circRNAs and their linear counterparts without or with internal introns. These circRNAs are C2A-2B-3-4, C2B-3-4 and C3-4, formed by the first four internal exons of SMN. When intercalating introns were encompassed in the constructs, augmented circRNA expression levels were perceived compared to intronless ones, suggesting a stimulatory effect of internal introns on backsplicing to generate SMN circRNAs. The findings also showed that SMN circRNAs were primarily localized in the cytoplasm. Subsequently, I am interested in uncovering the functional roles of SMN circRNAs. Through mechanisms including sponging/decoying miRNAs, interaction with RNA-binding proteins, and serving as translation templates, circRNAs regulate protein expressions and activities. Finally, the proteomics profile of the circRNA-overexpression cell lines was investigated via label-free LC-MS/MS quantification and bioinformatics analysis and validated our findings via qPCR and western blot. We identified several lead proteins associated with neuropathology, neurodevelopment, tumorigenesis, and spermatogenesis. These findings revealed the remarkable diversity of SMN circRNAs and provided pivotal insight into understanding the mechanism of their biogenesis and functions. This study opens novel avenues to exploit SMN-related applications, even beyond the scope of SMA.
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Type
dissertation