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

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.

Keywords: Elavl4 KO; HuD; alternative polyadenylation; alternative splicing; neocortex.

Figure 1

Alternative splicing (AS) events associated with deletion of HuD. (A) Total number of significantly different AS events in HuD KO cortices (n = 3). (B) Proportion of AS differences between KO and controls. (C) Number of increased and decreased inclusion AS levels in HuD KOs. (D) Top biological pathways associated with AS transcripts in HuD KO cortex analyzed by Ingenuity Pathway Analysis (IPA). Yellow line indicates p = 0.05. (E) Top neuronal functions affected by alternative splicing of transcripts. Blue lines predict inhibition of the function, while orange lines predict activation. Blue molecules indicate increased exon inclusion in HuD KOs, while red molecules indicate decreased exon inclusion.

Figure 2

Exon skipping (ES) events associated with HuD KO. (A) Volcano plot showing significant changes (−log(FDR)) vs. inclusion level (Inc level) difference (ΔPSI) between HuD KO and control mice. Blue dots show genes with significantly increased inclusion level differences and red dots show those with significantly decreased inclusion levels in HuD KO cortices. The most significant changes are identified by gene name (n = 3). (B) Top panels show sashimi plots demonstrating exon 2 skipping in the Elavl4 transcript, which is the exon deleted in HuD KOs. The bottom panel shows read coverage using IGV confirming exon 2 skipping in HuD KO. (C) Sashimi plots depicting exon 10 skipping and read coverage for exon 10 in Ap4e1 and (D) sashimi plots and exon 7 coverage in Rapgef4 in HuD KO and control mice.

Figure 3

List of transcripts that are alternatively spliced and directly interact with HuD. (A) Venn diagram showing the number of transcripts that are HuD targets and alternative spliced in HuD KO. List of genes with exon skipping (ES), alternative 3′ splice sites (A3SS) and alternative 5′ splice sites (A5SS) in HuD KO cortex. A negative inclusion level difference denotes an exon that is more excluded in KOs relative to controls, while a positive value indicates an exon with greater inclusion in HuD KO (n = 3). (B) Sashimi plots and read coverage of exon 3 in the Cbx3 transcript. This exon is the top included exon in KO mice. (C) Sashimi plots and read coverage of exon 12 in the Cspp1 transcript. This exon is the top excluded exon in KO mice.

Figure 4

Alternative splicing (AS) of Snap25and Gria2 transcripts in HuD KO cortex. (A) Snap25 sashimi plot depicting decreased exon 5b skipping in HuD KOs (n = 3). (B) Diagram showing AS of exons 5a and 5b in Snap25. (C) Read coverage of exon 5b using IGV and amino acid sequence comparison of exons 5a and 5b. (D) Gria2 sashimi plot depicting increased exon 14 skipping in HuD KO cortex (n = 3). (E) Diagram showing AS of the “flip or flop” isoforms of Gria2. (F) Read coverage of exon 14 using IGV and amino acid sequence comparison of exons 14 and 15.

Figure 5

No significant changes in the overall levels of mRNAs that show significant alterations in exon skipping in HuD KO cortex. Panels show the results of RNA-seq levels as log₂FPKM along with expression level p-values for six mRNAs that showed significant changes in exon skipping in HuD KO cortices (n = 3).

Figure 6

Differences in alternative polyadenylation (APA) of transcripts in HuD KO cortices. (A) Scatter plot depicting the percent distal usage index (PDUI) in control and HuD KO mice. Blue points represent significantly lengthened transcripts in HuD KOs, while red points represent significantly shortened transcripts (n = 3). (B) Complete list of lengthened and shortened transcripts in HuD KO cortex. (C) Biological pathways significantly enriched with alternatively polyadenylated transcripts identified by IPA. (D) Top neuronal functions affected by APA. Blue lines predict inhibition of the function, while orange lines predict activation. Blue molecules represent increased PDUI in HuD KOs, while red molecules represent decreased PDUI. (E) Read coverage graphs of the 3′-UTRs of Dtnbp1 and (F) Baiap2. Both transcripts exhibit decreased PDUI and shorter 3′-UTRs. Arrows show the position of the DaPars predicted proximal poly(A) signal (PAS) relative to the last coding exon.

Figure 7

Alternative polyadenylation of transcripts that directly interact with HuD. (A) Venn diagram and list of HuD targets that are alternatively polyadenylated in HuD KO mice. The 3′-UTR of Alg6 is lengthened in HuD KO cortex, while the 3′-UTRs of Max and Mmachc are shortened (n = 3). (B–D) Read coverage graphs of Alg6 3′-UTR, (B) Mmachc 3′-UTR (C) and Max 3′-UTR (D). Arrows indicate the position of the DaPars-predicted proximal PAS. For the Max transcript, the proximal PAS occurs in an intron.

Figure 8

No significant changes in the overall levels of mRNAs that showed significant alternative polyadenylation in HuD KO cortices. Panels show the results of RNA-seq levels as log₂FPKM along with p-values for five mRNAs that showed significant changes in alternative polyadenylation in HuD KO mice (n = 3). *** The significant decreases in Elavl4 mRNA levels in HuD KO mice are shown as a comparison set (n = 3).