Intron retention and nuclear loss of SFPQ are molecular hallmarks of ALS

Raphaelle Luisier,Bilal Malik,Linda Greensmith,Claire E Hall,Jernej Ule,Jia Newcombe,Nicholas M Luscombe,Helen Devine,Jamie S Mitchell,Ione Meyer,Giulia E Tyzack,Doaa M Taha,Rickie Patani

doi:10.1038/s41467-018-04373-8

Abstract

Mutations causing amyotrophic lateral sclerosis (ALS) strongly implicate ubiquitously expressed regulators of RNA processing. To understand the molecular impact of ALS-causing mutations on neuronal development and disease, we analysed transcriptomes during in vitro differentiation of motor neurons (MNs) from human control and patient-specific VCP mutant induced-pluripotent stem cells (iPSCs). We identify increased intron retention (IR) as a dominant feature of the splicing programme during early neural differentiation. Importantly, IR occurs prematurely in VCP mutant cultures compared with control counterparts. These aberrant IR events are also seen in independent RNAseq data sets from SOD1- and FUS-mutant MNs. The most significant IR is seen in the SFPQ transcript. The SFPQ protein binds extensively to its retained intron, exhibits lower nuclear abundance in VCP mutant cultures and is lost from nuclei of MNs in mouse models and human sporadic ALS. Collectively, we demonstrate SFPQ IR and nuclear loss as molecular hallmarks of familial and sporadic ALS.

Highlights

Mutations causing amyotrophic lateral sclerosis (ALS) strongly implicate ubiquitously expressed regulators of RNA processing
alternative polyadenylation (APA) is an alternative mode of RNA processing that generates distinct 3′ termini most frequently in the 3′ untranslated region (3′ UTR) of mRNA, thereby engendering isoforms of variable 3′ UTR length. 3′ UTRs serve as a key platform in the RNA regulatory network controlling mRNA translational efficiency, localisation and stability9,10. 3′ UTRs harbour extensive tissuespecific length variability that significantly affect their function[11]
To examine post-transcriptional changes during human motor neurogenesis, we analysed high-throughput RNA-sequencing (RNA-seq) data for polyadenylated RNA isolated from inducedpluripotent stem cells, neural precursors (NPCs; day 7), “patterned” precursor motor neurons (MNs), post-mitotic but electrophysiologically immature MNs (MNs; day 21), and electrophysiologically mature MNs derived from two patients with the ALS-causing valosin containing protein (VCP) gene mutation and two healthy controls (Fig. 1a; 31 samples from 5 time-points and 3 genotypes; 2 clones from 2 healthy controls and 3 clones from 2 ALS patients with VCP mutations: R155C and R191Q)

Summary

Introduction

Mutations causing amyotrophic lateral sclerosis (ALS) strongly implicate ubiquitously expressed regulators of RNA processing. ALScausing mutations implicate crucial regulators of RNA processing —normally expressed throughout development—in the underlying pathogenesis This raises the possibility that posttranscriptional changes occurring at early stages of life, including neurodevelopment, may play a pivotal role in the underlying molecular pathogenesis of ALS. Understanding the impact of ALS-causing mutations on neurodevelopment and disease will allow us to elucidate initiating molecular events This in turn may guide the development of new therapies targeting primary mechanisms before the disease progresses too far. Mutations perturbing the mRNA secondary structure and sites of mRNA–miRNA interactions can lead to neurodegeneration[14] Despite these findings, the roles of IR and 3′ UTR regulation in the context of MN development and homoeostasis have remained understudied compared with other forms of AS. Mutations in several RBPs including Transactive-response DNAbinding Protein, 43 kDa (TDP-43), Fused in Sarcoma (FUS) and TATA-Box Binding Protein Associated Factor 15 (TAF15) have been causally linked to familial form of ALS leading to RNA processing defects in mouse models[17,18]

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