MUTACLASH: identifying functional small RNA target sites using crosslinking-induced mutations

TL;DR

The paper introduces MUTACLASH, a tool that uses crosslinking-induced mutations to identify functional small RNA target sites in gene regulation.

Contribution

MUTACLASH is a novel bioinformatics tool that leverages CIMs to detect functional small RNA binding sites in CLASH data.

Findings

CIMs are enriched at the center of small RNA binding sites and at mismatched nucleotides in piRNA interactions.

mRNAs with CIMs show stronger regulatory effects than those without, even for noncanonical binding sites.

CIM analysis can identify functional small RNA binding sites missed by current tools.

Abstract

Small RNAs play essential roles in gene regulation across diverse biological processes. Crosslinking, ligation, and sequencing of hybrids (CLASH) experiments have revealed that PIWI and Argonaute proteins can each bind a wide range of mRNA targets with distinct base-pairing rules, raising questions about the flexibility and functional relevance of these interactions. Given that crosslinking-induced mutations (CIMs) provide single-nucleotide resolution molecular footprints of RNA-binding proteins, we developed MUTACLASH, a bioinformatics tool for systematically analyzing CIMs in CLASH data sets. Our analyses indicate that CIMs function as molecular footprints of Argonaute binding on target mRNAs. Specifically, for Caenorhabditis elegans miRNA and piRNA CLASH data, CIMs are enriched at the center of small RNA binding sites, as well as at nucleotides within mRNA target sites that exhibit local mismatches in piRNA interactions. Furthermore, we show that mRNAs with noncanonical miRNA and piRNA binding sites and/or low hybrid abundance marked by CIMs exhibit stronger regulatory effects than those without CIMs, demonstrating the utility of CIM analysis in identifying functional small RNA binding sites, including those that are otherwise likely overlooked with current analysis tools.

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