Alu insertion-mediated dsRNA structure formation with pre-existing Alu elements as a novel disease-causing mechanism

Abstract

We previously identified a homozygous Alu insertion variant (Alu_Ins) in the 3-UTR of the SPINK1 gene as the cause of a novel pediatric disease. Although we established that Alu_Ins leads to the complete loss of SPINK1 mRNA expression, the precise mechanisms remained elusive. Here we aimed to elucidate these mechanisms through a hypothesis-driven approach. Initially, we speculated that Alu_Ins could independently disrupt mRNA 3 end formation and/or affect other post-transcriptional processes such as nuclear export and translation, due to its particular location. However, the presence of Alu_Ins in the 3-UTR resulted in only an ~50% reduction in luciferase reporter activity compared to the wild-type, suggesting the involvement of additional mechanisms. Using RepeatMasker, we identified two Alu elements within SPINK1s third intron, both of which resided in an orientation opposite to that of Alu_Ins. Through RNAfold predictions and full-length gene expression assays designed to examine orientation-dependent interactions between Alu repeats, we present evidence linking the detrimental effect of Alu_Ins to extensive double-stranded RNA structures formed between Alu_Ins and pre-existing intronic Alu sequences. Our results reveal a novel pathogenetic mechanism involving an Alu insertion, highlighting the importance of considering interactions between new and pre-existing Alu elements in inverted orientations within disease-associated genes.