The RNA-Binding Protein HuD Regulates Alternative Splicing and Alternative Polyadenylation in the Mouse Neocortex.

Rebecca M Sena,Amy S Gardiner,Nora I Perrone-Bizzozero,David N Linsenbardt,Michela Dell'Orco,Jeffery L Twiss

doi:10.3390/molecules26102836

Rebecca M Sena, Amy S Gardiner + Show 4 more

Open Access

https://doi.org/10.3390/molecules26102836

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Abstract

The neuronal Hu/ELAV-like proteins HuB, HuC and HuD are a class of RNA-binding proteins that are crucial for proper development and maintenance of the nervous system. These proteins bind to AU-rich elements (AREs) in the untranslated regions (3′-UTRs) of target mRNAs regulating mRNA stability, transport and translation. In addition to these cytoplasmic functions, Hu proteins have been implicated in alternative splicing and alternative polyadenylation in the nucleus. The purpose of this study was to identify transcriptome-wide effects of HuD deletion on both of these nuclear events using RNA sequencing data obtained from the neocortex of Elavl4–/– (HuD KO) mice. HuD KO affected alternative splicing of 310 genes, including 17 validated HuD targets such as Cbx3, Cspp1, Snap25 and Gria2. In addition, deletion of HuD affected polyadenylation of 53 genes, with the majority of significantly altered mRNAs shifting towards usage of proximal polyadenylation signals (PAS), resulting in shorter 3′-UTRs. None of these genes overlapped with those showing alternative splicing events. Overall, HuD KO had a greater effect on alternative splicing than polyadenylation, with many of the affected genes implicated in several neuronal functions and neuropsychiatric disorders.

Highlights

RNA-binding proteins (RBPs) serve important functions in the co-transcriptional and post-transcriptional control of gene expression [1,2]
Exon skipping represented the largest proportion of alternative splicing between groups at 77.74%, while both intron retention and mutually exclusive exons represented 3.55% (Figure 1B)
To identify genes that were directly affected by KO of this protein, we focused only on transcripts that had been shown to directly bind to HuD by RNA immunoprecipitation (RIP) assays

Summary

Introduction

RNA-binding proteins (RBPs) serve important functions in the co-transcriptional and post-transcriptional control of gene expression [1,2]. It has been estimated that 92–95% of human genes undergo alternative splicing to produce multiple protein isoforms from one gene [3,4] These events occur at the highest frequencies in the brain, with neuron development and function impacted [5]. A large proportion of mRNA diversity is attributed to polyadenylation, and a large number of transcripts in the nervous system possess longer 3 -UTRs [11,12,13] These longer 3 -UTR isoforms mediate mRNA localization to dendrites and axons and can be subjected to RBP and microRNA-mediated decay in the cell [12]. We sought to examine the effects of HuD deletion on these processes in the neocortex of adult Elavl4–/– (HuD KO) mice

Alternative Splicing of Transcripts in HuD KO Cortex

Animals

Quality Check of Sequencing Reads and Alignment