Identification of RBMX as a splicing regulator in Parkinsonian mimetic induced alternative splicing of α-synuclein

Abstract

α-Synuclein (α-syn) plays a precipitating role in Parkinson's disease (PD) due to its tendency to form oligomers and fibrils. The presence of smaller isoforms of α-syn was widely noticed in the affected brain regions of PD patients. 112-synuclein (112-syn) which lacks exon-5, possess enhanced aggregation propensity and forms intracellular inclusions. However, the factors responsible for the skipping of exon-5 are not completely understood. In this context, we aimed to identity the cis & trans-acting elements governing alternative splicing (AS) events by the Parkinsonian agent (MPP+) using minigene constructs. Minigene-I and -II were constructed by pruning the intron-4 and -5 regions respectively without altering the branch point adenosine to preserve splicing machinery. Also, chimeric minigenes were engineered by replacing either 5′ (Mini-III) or 3′ (Mini-IV) flanking intronic regions of exon-5 with other intronic regions (intron-3 and -2) that are not responsive to MPP+ induced splicing. While all the above minigenes exhibited MPP+-induced skipping of exon-5, Minigene-III did not generate the spliced product indicating that the 5′ flanking intronic region (316 bp) of exon-5 possess cis-acting elements responsible for oxidant-induced alternative splicing. RNA-Binding Protein Database (RBDP) analysis revealed the presence of four putative RNA binding proteins (RBPs), namely, RBMX, MBNL1, KHDRBS3 and SFRS1 that may bind to the 316 bp region of intron-4and their expression was substantially reduced following MPP+ treatment. Further, overexpression of RBMX mitigated MPP+-induced generation of 112-syn and also reduced intracellular α-syn aggregates. Overall, our study identified the pivotal role of the splicing regulator, RBMX, in the pathophysiology of PD.