The World of Stable Ribonucleoproteins and Its Mapping With Grad-Seq and Related Approaches.

Jörg Vogel,Alexandre Smirnov,Milan Gerovac

doi:10.3389/fmolb.2021.661448

Abstract

Macromolecular complexes of proteins and RNAs are essential building blocks of cells. These stable supramolecular particles can be viewed as minimal biochemical units whose structural organization, i.e., the way the RNA and the protein interact with each other, is directly linked to their biological function. Whether those are dynamic regulatory ribonucleoproteins (RNPs) or integrated molecular machines involved in gene expression, the comprehensive knowledge of these units is critical to our understanding of key molecular mechanisms and cell physiology phenomena. Such is the goal of diverse complexomic approaches and in particular of the recently developed gradient profiling by sequencing (Grad-seq). By separating cellular protein and RNA complexes on a density gradient and quantifying their distributions genome-wide by mass spectrometry and deep sequencing, Grad-seq charts global landscapes of native macromolecular assemblies. In this review, we propose a function-based ontology of stable RNPs and discuss how Grad-seq and related approaches transformed our perspective of bacterial and eukaryotic ribonucleoproteins by guiding the discovery of new RNA-binding proteins and unusual classes of noncoding RNAs. We highlight some methodological aspects and developments that permit to further boost the power of this technique and to look for exciting new biology in understudied and challenging biological models.

Highlights

When about two decades ago high-throughput approaches in molecular biology became a reality, it all began with parts lists
Genome-wide functional screens were greatly facilitated by modern mutagenesis tools based on random transposon insertion and CRISPR-mediated gene disruption, silencing, or activation (Ford et al, 2019; Cain et al, 2020; Jiang et al, 2020)
Many techniques have been developed to identify partners of specific RNAs and RNA-binding proteins (RBPs) with mass spectrometry-based proteomics and RNAseq, respectively (Saliba et al, 2017; Giambruno et al, 2018; Lin and Miles, 2019). Among the latter, crosslinking approaches based on the covalent fixation of direct and often transitory RNA–protein associations, such as CLIP-seq, enjoy the widest popularity (Andresen and Holmqvist, 2018; Lee and Ule, 2018; Tree et al, 2018)

Summary

INTRODUCTION

When about two decades ago high-throughput approaches in molecular biology became a reality, it all began with parts lists. Many techniques have been developed to identify partners of specific RNAs and RNA-binding proteins (RBPs) with mass spectrometry-based proteomics and RNAseq, respectively (Saliba et al, 2017; Giambruno et al, 2018; Lin and Miles, 2019) Among the latter, crosslinking approaches based on the covalent fixation of direct and often transitory RNA–protein associations, such as CLIP-seq, enjoy the widest popularity (Andresen and Holmqvist, 2018; Lee and Ule, 2018; Tree et al, 2018). The time has come to compile the next-level parts list—that of ribonucleoprotein complexes (RNPs)

WHAT IS A STABLE RNP?

ONTOLOGY OF STABLE RNPS

PROFILING CELLULAR COMPLEXES WITH COMPLEXOMIC TECHNIQUES

INCORPORATING RNA IN THE COMPLEXOME LANDSCAPE

General Pipeline