The kinetic landscape of an RNA-binding protein in cells

Abstract

Gene expression in higher eukaryotic cells orchestrates interactions between thousands of RNA binding proteins (RBPs) and tens of thousands of RNAs 1 . The kinetics by which RBPs bind to and dissociate from their RNA sites are critical for the coordination of cellular RNA-protein interactions 2 . However, these kinetic parameters were experimentally inaccessible in cells. Here we show that time-resolved RNA-protein crosslinking with a pulsed femtosecond UV laser, followed by immunoprecipitation and high throughput sequencing allows the determination of binding and dissociation kinetics of the RBP Dazl for thousands of individual RNA binding sites in cells. This kinetic crosslinking and immunoprecipitation (KIN-CLIP) approach reveals that Dazl resides at individual binding sites only seconds or shorter, while the sites remain Dazl-free markedly longer. The data further indicate that Dazl binds to many RNAs in clusters of multiple proximal sites. The impact of Dazl on mRNA levels and ribosome association correlates with the cumulative probability of Dazl binding in these clusters. Integrating kinetic data with mRNA features quantitatively connects Dazl-RNA binding to Dazl function. Our results show how kinetic parameters for RNA-protein interactions in cells can be measured and how these data quantitatively link RBP-RNA binding to cellular RBP function.

Multifunctional RNA-binding proteins influence mRNA abundance and translational efficiency of distinct sets of target genes

“…Mechanistic insight into the quantitative effects of RBP expression on mRNA translation - i.e., when translation rates of the endogenous RBP targets respond directly to changes in RBP abundance - was recently provided by several studies (Chothani et al, 2019; Luo et al, 2020; Sharma et al, 2021). These studies used this quantitative relationship to assign novel functions to known RBPs and to investigate the kinetics through which RBPs regulate their targets.…”

Rapidly Growing Protein-Centric Technologies to Extensively Identify Protein–RNA Interactions: Application to the Analysis of Co-Transcriptional RNA Processing

“…Recently, a pulsed femtosecond UV to CLIP methodology (kinetic cross-linking and immunoprecipitation; KIN-CLIP) has been reported [ 59 ]. Compared with conventional UV irradiation, high-intensity UV irradiation markedly increases the crosslinking efficiency without altering the RNA–protein crosslinking patterns.…”

Oxidative Modifications of RNA and Its Potential Roles in Biosystem

“…There have been numerous studies regarding RNA-protein artificial cross-linking, mainly for mapping between RNA and its associated RNA binding proteins by means of site-specific probe insertion and UV irradiation ( i.e. ( Van Nostrand et al, 2016 ; Lin and Miles, 2019 ; Sharma et al, 2021 )). For pathophysiological cross-linking, Mirzaei and Regnier reported RNA and protein cross-linking in yeast after H 2 O 2 treatment ( Mirzaei and Regnier, 2006 ).…”