Transcriptome-wide alternative mRNA splicing analysis reveals post-transcriptional regulation of neuronal differentiation

Abstract

Alternative splicing (AS) plays an important role in neuronal development, function, and disease. Efforts to analyze the transcriptome of AS in neurons on a wide scale are currently limited. We characterized the transcriptome-wide AS changes in SH-SY5Y neuronal differentiation model, which is widely used to study neuronal function and disorders. Our analysis revealed global changes in five AS programs that drive neuronal differentiation. Motif analysis revealed the contribution of RNA-binding proteins (RBPs) to the regulation of AS during neuronal development. We concentrated on the primary alternative splicing program that occurs during differentiation, specifically on events involving exon skipping (SE). Motif analysis revealed motifs for polypyrimidine tract-binding protein 1 (PTB) and ELAV-like RNA binding protein 1 (HuR/ELAVL1) to be the top enriched in SE events, and their protein levels were downregulated after differentiation. shRNA knockdown of either PTB and HuR was associated with enhanced neuronal differentiation and transcriptome-wide exon skipping events that drive the process of differentiation. At the level of gene expression, we observed only modest changes, indicating predominant post-transcriptional effects of PTB and HuR. We also observed that both RBPs altered cellular responses to oxidative stress, in line with the differentiated phenotype observed after either gene knockdown. Our work characterizes the AS changes in a widely used and important model of neuronal development and neuroscience research and reveals intricate post-transcriptional regulation of neuronal differentiation.

Keywords: PTB, HuR; SH‐SY5Y; alternative splicing; neurodegeneration; neuronal differentiation.