Transcriptome- and proteome-wide effects of circular RNAs of SMA genes - Project Summary: Spinal Muscular Atrophy (SMA), the leading genetic cause of infant mortality, results from deficiency of Survival Motor Neuron (SMN) protein due to deletions of or mutations in the SMN1 gene. SMN2, a near identical copy of SMN1, fails to compensate for the loss of SMN1 due to predominant skipping of exon 7. SMN1/2 harbor unusually high numbers of Alu elements that are often associated with the generation of circular RNAs (circRNAs). Consistently, we and others have reported a huge repertoire of circRNAs produced by SMN1/2. C2A-2B-3-4, C2B-3-4 and C3-4 encompassing early exons of SMN1/2 are the most abundantly- expressed SMN1/2 circRNAs. We previously reported that C2A-2B-3-4 is downregulated in type 1 SMA patient cells, and its expression is cross-regulated by C2B-3-4 and C3-4. A recent report showed a correlation between overexpression of C2B-3-4 and improved motor outcomes in type I SMA patients treatment with Nusinersen, an antisense oligonucleotide-based drug. These findings underscore SMN1/2 circRNAs as novel candidates for diagnosis and therapy. Yet, very little is known about the functions of SMN1/2 circRNAs. We recently performed transcriptome and proteome analysis of inducible HEK293 cells stably overexpressing C2A-2B-3-4. Our results captured the altered expression of 4172 genes and 118 proteins. Surprisingly, we also observed chromosome-specific effects as expression of many genes located on chromosomes 4, 7, 10 and X were specifically impacted. In this proposal, we will perform similar transcriptome and proteome analysis of inducible HEK293 cells stably overexpressing C2B-3-4 and C3-4. We will expand this study to examine the effect of the transient overexpression of C2A-2B-3-4, C2B-3-4 and C3-4 in different cell types, including HeLa, neuronal SH-SY5Y, motor neuron-like NSC-34 cells as well as in SMA patient fibroblasts. We will also examine the effect of depletion of C2A-2B-3-4, C2B-3-4 and C3-4 on the transcriptome and proteome in different cell types. We will independently validate the findings of transcriptomic and proteomic data by qPCR and western blot, respectively. Using gel-based assays, we will validate the effects of C2A-2B-3-4, C2B-3-4 and C3-4 on splicing events. We will perform computational analysis to uncover potential miRNAs that are likely to be sponged by SMN1/2 circRNAs and affect translation of the target proteins. We will investigate if specific promoters and/or chromatin modifications are direct targets of C2A-2B-3-4, C2B-3-4 and C3-4. Findings will uncover SMN protein-independent functions of SMN1/2 in diverse cellular processes, including DNA replication, DNA repair, transcription, splicing, translation, cell signaling, macromolecular trafficking, stress granule formation, mitochondrial regeneration, and cytoskeletal dynamics. Importantly, results will reveal common as well as distinct functions of SMN1/2 circRNAs differing by one or two early exons of SMN1/2. Outcomes will significantly advance our understanding of SMA, and possibly many other diseases associated with the aberrant expression of SMN1/2 circRNAs.
