Selection preserves Ubiquitin Specific Protease 4 alternative exon skipping in therian mammals.

Caitlyn Vlasschaert,Douglas A Gray,Xuhua Xia

doi:10.1038/srep20039

Abstract

Ubiquitin specific protease 4 (USP4) is a highly networked deubiquitinating enzyme with reported roles in cancer, innate immunity and RNA splicing. In mammals it has two dominant isoforms arising from inclusion or skipping of exon 7 (E7). We evaluated two plausible mechanisms for the generation of these isoforms: (A) E7 skipping due to a long upstream intron and (B) E7 skipping due to inefficient 5′ splice sites (5′SS) and/or branchpoint sites (BPS). We then assessed whether E7 alternative splicing is maintained by selective pressure or arose from genetic drift. Both transcript variants were generated from a USP4-E7 minigene construct with short flanking introns, an observation consistent with the second mechanism whereby differential splice signal strengths are the basis of E7 skipping. Optimization of the downstream 5′SS eliminated E7 skipping. Experimental validation of the correlation between 5′SS identity and exon skipping in vertebrates pinpointed the +6 site as the key splicing determinant. Therian mammals invariably display a 5′SS configuration favouring alternative splicing and the resulting isoforms have distinct subcellular localizations. We conclude that alternative splicing of mammalian USP4 is under selective maintenance and that long and short USP4 isoforms may target substrates in various cellular compartments.

Highlights

Ubiquitin-specific protease 4 (USP4) is a deubiquitinating enzyme that can edit or remove ubiquitin chains of various topologies
It has been proposed that mutations that weaken the 5′ splice site are responsible for the evolutionary shift from constitutive to alternative splicing in many vertebrate genes, as reviewed in Keren et al.[21], and compelling evidence has been presented in support of this hypothesis[34]
While most minor splice variants are attributable to noisy splicing[35,36], Ubiquitin specific protease 4 (USP4) constitutes a rare case wherein selective pressure acts to conserve differential 5′ splice site (SS) strengths leading to exon skipping in therian mammals

Summary

Introduction

Ubiquitin-specific protease 4 (USP4) is a deubiquitinating enzyme that can edit or remove ubiquitin chains of various topologies. Despite the proposed facultative role of this alternatively spliced exon[16], the majority of conserved phosphorylation sites in USP4 are within E7, suggesting the protein isoforms may be differently regulated[17]. The alternative inclusion of E7 in USP4 mRNA could be explained by appropriately long flanking introns (Fig. 1A(ii)) We evaluated these alternative hypotheses by constructing a computational evolutionary framework to characterize USP4 splicing patterns in multiple vertebrate lineages and tested our in silico predictions in the laboratory to gauge whether the two isoforms of USP4 observed in humans have discrete functional and/or regulatory roles or whether they are the by-product of reduced selection for E7 retention

Methods

Results

Conclusion