Origin of a novel CYP20A1 transcript isoform through multiple Alu exaptations creates a potential miRNA sponge

Abstract

Background: Primate-specific Alus contribute to transcriptional novelties in conserved gene regulatory networks. Alu RNAs are present at elevated levels in stress conditions and consequently leads to transcript isoform specific functional role modulating the physiological outcome. One of the possible mechanisms could be Alu nucleated mRNA-miRNA interplay.Result: Using combination of bioinformatics and experiments, we report a transcript isoform of an orphan gene, CYP20A1 (CYP20A1_Alu-LT) through exaptation of 23 Alus in its 9kb 3'UTR. CYP20A1_Alu-LT, confirmed by 3'RACE, is an outlier in length and expressed in multiple cell lines. We demonstrate its presence in single nucleus RNA-seq of ~16000 human cortical neurons (including rosehip neurons). Its expression is restricted to the higher primates. Most strikingly, miRanda predicts ~4700 miRNA recognition elements (MREs; with threshold< -25kcal/mol) for ~1000 miRNAs, which have majorly originated within the 3'UTR-Alus post exaptation. We hypothesized that differential expression of this transcript could modulate mRNA-miRNA networks and tested it in primary human neurons where CYP20A1_Alu-LT is downregulated during heat shock response and upregulated upon HIV1-Tat treatment. CYP20A1_Alu-LT could possibly function as a miRNA sponge as it exhibits features of a sponge RNA such as cytosolic localization and ≥10 MREs for 140 miRNAs. Small RNA-seq revealed expression of nine miRNAs that can potentially be sponged by CYP20A1_Alu-LT in neurons. Additionally, CYP20A1_Alu-LT expression was positively correlated (low in heat shock and high in Tat) with 380 differentially expressed genes that contain cognate MREs for these nine miRNAs. This set is enriched in genes involved in neuronal development and hemostasis pathways. Conclusion:We demonstrate a potential role for CYP20A1_Alu-LT as miRNA sponge through preferential presence of MREs within Alus in a transcript isoform specific manner. This highlights a novel component of Alu-miRNA mediated transcriptional modulation leading to physiological homeostasis.Nearly 14% of the human transcriptome contain exonized Alus, predominantly in the principal isoforms of genes. Exonization is frequently reported in genes that have arisen de novo in primates, with most of the events in 3'UTRs (18,19). Such exonized Alus can increase the regulatory possibilities for a transcript, in a spatio-temporal manner, through antisense, miRNAs, A-to-I RNA editing, alternative splicing and enhancers. We have shown that transcript isoform dynamics i.e., the relative proportion of exonized versus non-exonized isoforms of a gene, could be modulated by these events in viral recovery and stress response (19,20). Besides, Alus provide substrates for other regulatory events such as gain of poly-A sites, AU-rich motifs and miRNA recognition elements (MREs) that can result in alternative polyadenylation, mRNA decay or translation stalling; and formation of specific secondary structures (9,(21)(22)(23)(24)(25). We have also reported that Alu-MREs can alter trans...