Novel autoregulatory cases of alternative splicing coupled with nonsense-mediated mRNA decay

Abstract

Nonsense-mediated decay (NMD) is a eukaryotic mRNA surveillance system that selectively degrades 1 transcripts with premature termination codons (PTC). Many RNA-binding proteins (RBP) regulate their 2 expression levels by a negative feedback loop, in which RBP binds its own pre-mRNA and causes alternative 3 splicing to introduce a PTC. We present a bioinformatic framework to identify novel such autoregulatory 4 feedback loops by combining eCLIP assays for a large panel of RBPs with the data on shRNA inactivation 5 of NMD pathway, and shRNA-depletion of RBPs followed by RNA-seq. We show that RBPs frequently bind 6 their own pre-mRNAs and respond prominently to NMD pathway disruption. Poison and essential exons, 7 i.e., exons that trigger NMD when included in the mRNA or skipped, respectively, respond oppositely to the 8 inactivation of NMD pathway and to the depletion of their host genes, which allows identification of novel 9 autoregulatory mechanisms for a number of human RBPs. For example, SRSF7 binds its own pre-mRNA 10 and facilitates the inclusion of two poison exons; SFPQ binding promotes switching to an alternative distal 11 3'-UTR that is targeted by NMD; RPS3 activates a poison 5'-splice site in its pre-mRNA that leads to a 12 frame shift; U2AF1 binding activates one of its two mutually exclusive exons, leading to NMD; TBRG4 is 13 regulated by cluster splicing of its two essential exons. Our results indicate that autoregulatory negative 14 feedback loop of alternative splicing and NMD is a generic form of post-transcriptional control of gene 15 expression. 16Introduction 17 Gene expression in higher eukaryotes is regulated at many different levels. The output of the transcriptional 18 program is maintained by a large number of protein factors and cis-regulatory elements, which control 19 the balance between mRNA production and degradation [1,2]. Nonsense mutations and frame-shifting 20 splicing errors induce premature termination codons (PTC) that give rise to mRNAs encoding truncated, 21 dysfunctional proteins. In eukaryotic cells, mRNA transcripts with PTC are selectively degraded by the 22 surveillance mechanism called Nonsense-Mediated mRNA Decay (NMD) [3]. 23The so-called exon junction complex-dependent (EJC) model postulates that NMD distinguishes between 24 normal and premature translation termination in the cytoplasm, where ribosomes displace EJCs from within, 25 but not downstream of the reading frame [4,5]. These complexes are deposited approximately 50 nucleotides 26 (nt) upstream of the exon-exon junctions during pre-mRNA splicing. EJCs that remain associated with 27Short-hairpin RNA knockdown of RBP followed by RNA-seq 131 Publicly available data on short-hairpin (shRNA) knockdown of 250 RBPs followed by RNA-seq (shRNA-132 KD) [51] were downloaded in BAM format from ENCODE data repository [52,53]. A summary of RBP 133 depletion data and the respective accession numbers is given in Supplementary Table S1. Exon inclusion 134 metrics (PSI) were called for all annotated exons using IPSA software...