Advantages and limitations of UV cross‐linking analysis of protein–RNA interactomes in microbes

Abstract

RNA‐binding proteins (RBPs) govern the lifespan of nearly all transcripts and play key roles in adaptive responses in microbes. A robust approach to examine protein–RNA interactions involves irradiating cells with UV light to form covalent adducts between RBPs and their cognate RNAs. Combined with RNA or protein purification, these procedures can provide global RBP censuses or transcriptomic maps for all target sequences of a single protein in living cells. The recent development of novel methods has quickly populated the RBP landscape in microorganisms. Here, we provide an overview of prominent UV cross‐linking techniques which have been applied to investigate RNA interactomes in microbes. By assessing their advantages and caveats, this technical evaluation intends to guide the selection of appropriate methods and experimental design as well as to encourage the use of complementary UV‐dependent techniques to inspect RNA‐binding activity.

ChimericFragments: computation, analysis and visualization of global RNA networks

“…The past few years have brought a revolution in our understanding of how RNA–RNA interactions form at a global level due to the development of various new sequencing-based technologies ( 7 , 9 , 10 ). In contrast to previous approaches, which frequently relied on the identification of individual RNA duplexes and/or the characterization of single RNA regulators, these technologies have paved the way to simultaneously analyze the interactomes of dozens to hundreds of regulatory RNAs and thousands of RNA–RNA pairs ( 7 ).…”

pyRBDome: A comprehensive computational platform for enhancing and interpreting RNA-binding proteome data

“…These methods usually involve UV or chemical treatment of cells to create covalent bonds between proteins and direct RNA substrates. This is followed by enrichment of the cross-linked RNA-protein complexes and identification of proteins by quantitative mass spectrometry (MS) (reviewed in (Esteban-Serna et al , 2023)). Common approaches for enriching RNA-protein complexes include using oligo(dT) beads to capture proteins cross-linked to polyadenylated RNAs (Castello et al , 2012, 2016; Baltz et al , 2012; Stenum et al , 2023), silica beads that capture all RNAs and cross-linked proteins (Asencio et al , 2018; Chu et al , 2022; Shchepachev et al , 2019; Trendel et al , 2019; Beckmann et al , 2015; Bae et al , 2020) or organic–aqueous phase separation methods that rely on the fact that cross-linked RNAs alter the physiochemical properties of proteins (Queiroz et al , 2019; Smith et al , 2020; Trendel et al , 2019; Urdaneta et al , 2019).…”