Discovering the 3′ UTR-mediated regulation of alpha-synuclein

12888–12903 Nucleic Acids Research, 2017, Vol. 45, No. 22 doi: 10.1093/nar/gkx1048 Published online 15 November 2017 Discovering the 3 UTR-mediated regulation of alpha-synuclein Domenica Marchese1,2 , Teresa Botta-Orfila1,2 , Davide Cirillo1,2,3 , Juan Antonio Rodriguez2,4 , Carmen Maria Livi1,2,5 , Rubén Fernández-Santiago6,7 , Mario Ezquerra6,7 , Maria J. Martı́6,7 , Elias Bechara1,2 and Gian Gaetano Tartaglia1,2,8,*,† 1 Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain, 2 Universitat Pompeu Fabra (UPF), Barcelona, Spain, 3 Barcelona Supercomputing Center (BSC), Torre Girona c/Jordi Girona, 29, 08034 Barcelona, Spain, 4 Centro Nacional de Análisis Genómico, c/BaldiriReixac, 4, 08028 Barcelona, Spain, 5 IFOM, the FIRC Institute of Molecular Oncology, Via Adamello 16, 20139 Milan, Italy, 6 Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain, 7 Parkinson’s Disease and Movement Disorders Unit, Institut de Neurociències Hospital Clı́nic, CIBERNED, Barcelona, Spain and 8 Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Received July 05, 2017; Revised October 05, 2017; Editorial Decision October 17, 2017; Accepted October 20, 2017 ABSTRACT Recent evidence indicates a link between Parkinson’s Disease (PD) and the expression of a-synuclein (SNCA) isoforms with different 3 untranslated regions (3 UTRs). Yet, the post-transcriptional mechanisms regulating SNCA expression are unknown. Using a large-scale in vitro /in silico screening we identified RNA-binding proteins (RBPs) that interact with SNCA 3 UTRs. We identified two RBPs, ELAVL1 and TIAR, that bind with high affinity to the most abundant and translationally active 3 UTR isoform (575 nt). Knockdown and overexpression experiments indicate that both ELAVL1 and TIAR positively regulate endogenous SNCA in vivo. The mechanism of regulation implies mRNA stabilization as well as enhancement of translation in the case of TIAR. We observed significant alteration of both TIAR and ELAVL1 expression in motor cortex of postmortem brain donors and primary cultured fibroblast from patients affected by PD and Multiple System Atrophy (MSA). Moreover, trans expression quantitative trait loci (trans-eQTLs) analysis revealed that a group of single nucleotide polymorphisms (SNPs) in TIAR genomic locus influences SNCA expression in two different brain areas, nucleus accumbens and hippocampus. Our study sheds light on the 3 UTRmediated regulation of SNCA and its link with PD pathogenesis, thus opening up new avenues for in- vestigation of post-transcriptional mechanisms in neurodegeneration. INTRODUCTION Parkinson’s disease (PD) is the second most common human neurodegenerative disorder, after Alzheimer disease. PD is a multifactorial disorder in which different factors such as aging, genetic susceptibility and environmental insults converge to cause neurodegeneration. Idiopathic PD represents over 90% of PD cases, while genetic PD, caused by mutations in one or more of the PD-associated loci, is only 10% of the cases (1). PD initiates in the central nervous system and spreads to the peripheral and enteric parts of the nervous system. The most important feature in the brains of PD patients is the selective loss of dopaminergic pigmented neurons within the substantia nigra and, to a lesser extent, neurons residing in the ventral tegmental and retrorubral areas (2). The neuropathological hallmark of Parkinson’s disease is the presence of eosinophilic inclusion in the soma of neurons known as Lewy bodies (LBs), as well as in the neurites where the inclusions are called Lewy neurites. LBs are composed of a mixture of lipids, neuromelanin and up to several hundred individual proteins, including ubiquitin, heatshock proteins, dj-1, sod1 and 2, synphilin-1, tau, tyrosine hydroxylase, and many others, but the key component is ␣-synuclein (SNCA gene), a small protein enriched at the presynaptic terminals (3). The post-transcriptional mechanisms controlling SNCA expression are at present unknown, although we previously observed that UTR-mediated regulation could play a key role in controlling ␣-synuclein protein abundance (4). * To whom correspondence should be addressed. Tel: +34 93 316 01 16; Fax: +34 93 396 99 83; Email: † On behalf of the Catalan MSA Registry (CMSAR)––see Appendix.  C The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Nucleic Acids Research, 2017, Vol. 45, No. 22 12889 Specific SNCA transcript isoforms with different 3 UTR lengths have been found enriched in cerebral cortex samples of post-mortem PD patients (5). Recently, in a cohort of 202 cases of de novo motor PD, significantly lower levels of SNCA mRNA with extended 3 UTRs have been quantified by digital expression analysis (6). These findings are particularly relevant to PD etiopathogenesis: a switch of alternative polyadenylation to favour expression of specific SNCA 3 UTR isoforms enables the binding of a number of transacting factors that alter protein production, localisation and function. Post-transcriptional networks induce changes in expression levels that are about one order of magnitude smaller than those caused by transcription factors, but ␣-synuclein is highly concentrated at the pre-synaptic terminals of neurons (70–140 uM) and even small fluctuations of its concentration can indeed induce aggregation (7). In the present work we address the question of which RNA-binding proteins (RBPs) are able to bind the different 3 UTRs of SNCA mRNA and control its expression. Following the unbiased discovery of protein interactors by means of a large-scale in vitro / in silico screening, we prioritised two RBPs, ELAVL1 and TIAR, that target SNCA 3 UTRs and their influence stability and translation efficiency. Our study suggests that TIAR and ELAVL1 are key regulators of ␣-synuclein intracellular concentration and function. MATERIALS AND METHODS Human protein array The RNA sequence corresponding to SNCA 3 UTR short (575 bp), medium (1.07 kb) and long (2.5 kb) were cloned in pBluescript-SK(+) empty plasmid. The RNA probes of the SNCA long 3 UTR (3 UTRL) were synthesized by in vitro transcription (IVT) with T7 and T3 RNA polymerase (Agilent) for the sense and antisense strand respectively, and fluorescently labeled with Label IT ␮Array Cy5 labeling kit (Mirus) applying minor modifications to manufacturer’s instructions. RNA concentration and labeling density were measured with Nanodrop 1000 spectrophotometer (Thermo Scientific). RNA integrity was veri (...truncated)