Protein–RNA interactions: from mass spectrometry to drug discovery

Abstract

Proteins and RNAs are fundamental parts of biological systems, and their interactions affect many essential cellular processes. Therefore, it is crucial to understand at a molecular and at a systems level how proteins and RNAs form complexes and mutually affect their functions. In the present mini-review, we will first provide an overview of different mass spectrometry (MS)-based methods to study the RNA-binding proteome (RBPome), most of which are based on photochemical cross-linking. As we will show, some of these methods are also able to provide higher-resolution information about binding sites, which are important for the structural characterisation of protein–RNA interactions. In addition, classical structural biology techniques such as nuclear magnetic resonance (NMR) spectroscopy and biophysical methods such as electron paramagnetic resonance (EPR) spectroscopy and fluorescence-based methods contribute to a detailed understanding of the interactions between these two classes of biomolecules. We will discuss the relevance of such interactions in the context of the formation of membrane-less organelles (MLOs) by liquid–liquid phase separation (LLPS) processes and their emerging importance as targets for drug discovery.

Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry

“…893 Recent technical advances also enabled the visualization of protein-RNA interaction. 894 Various XL chemistries are available (amine-reactive, sulfhydryl and photoreactive crosslinkers; reversible vs irreversible) and cross-linked proteins detected by mass spectrometry. 895 In general, applying XL-MS to a mixture of interacting, purified proteins is preferable to in situ XL of complex protein mixtures (i.e., cell lysate) as detection and deconvolution of XL peptides is technically and computationally challenging.…”

“…Chemical cross-linking (XL) of proteins can determine PPIs with amino-acid level resolution, and can thereby give valuable insights into the orientation of two or more proteins relative to one another . Recent technical advances also enabled the visualization of protein-RNA interaction . Various XL chemistries are available (amine-reactive, sulfhydryl and photoreactive crosslinkers; reversible vs irreversible) and cross-linked proteins detected by mass spectrometry .…”

A special issue ofEssays in Biochemistryon structural mass spectrometry

“…XL-MS technology has found broad application, including for the study of proteins/assemblies in vitro and in cell, but faces several analytical challenges as outlined here by Lee and O’Reilly [ 21 ]. Similarly, new advances are occurring to study protein–nucleic acid assemblies using XL-MS methodologies as described by Steinmetz et al [ 22 ].…”