The kinetic landscape of an RNA binding protein in cells

Abstract

4) Corresponding authors 2 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 RNAprotein interactions 2 . However, these kinetics 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 previously inaccessible, 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

“…Although the precise RBP mechanisms behind this dual functionality will require more follow-up, we discovered that the specific affinity of several RBPs to structural properties of mRNAs, such as protein-coding sequence length, UTR length or RNA secondary structure, contribute separately to the observed independent effects on mRNA abundance or translation. In support of our findings, a recent study (Sharma et al, 2021) has inspected the connection between the binding kinetics of the RBP DAZL and their effect on mRNA abundance and translation of specific sets of target genes, identifying several 3' UTR features -UTR length, presence of binding clusters, distance to the polyadenylation site -that are linked to the trait-specific regulation of different groups of targets. In addition, the usage of different ribosome binding domains, the recognition of alternative RBP motifs and the presence of binding sites located in different gene regions (i.e., UTRs, CDS, or introns) can be also indicative of RBP multi-functionality (Ray et al, 2013).…”

“…Here, we built an in-silico method for the large-scale analysis of RBP-driven regulation using correlation as a proxy for mRNA abundance and translational efficiency (TE) of target genes in the human heart. Our approach underscores the functional importance of RBP expression fluctuations in the control of gene expression, a mechanistic feature recently highlighted by others in vitro (Chothani et al, 2019;Luo et al, 2020;Sharma et al, 2021). We exploited the quantitative effect of RBPs on known target genes to implicate 74 RBPs in the regulation of mRNA abundance and translation.…”

“…During translation, RBPs can act in a general capacity (e.g., mRNA translation initiation or elongation), as well as in more specialized functions where select accessory RBPs or heterogeneous ribosomes facilitate the translation of dedicated subsets of target RNAs (Harvey et al, 2018;Shi et al, 2017;Simsek et al, 2017). 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 levels -was recently provided by several studies that used this relationship to assign novel functions to known RBPs (Chothani et al, 2019;Luo et al, 2020;Sharma et al, 2021) and to define the kinetics through which RBPs regulate their targets (Sharma et al, 2021). Chothani et al used a correlation-based approach, for which the frequency with which RBP levels correlate with target translation rates was used to pinpoint key RBP network hubs crucial for translational regulation during cardiac fibrosis (Chothani et al, 2019).…”

“…This resulted in the novel identification of the stress granule RBP UBAP2L as a ribosome-associated RBP (Luo et al, 2020). Lastly, Sharma et al developed a new approach to investigate RNA-protein kinetics in a time-resolved manner for the RBP DAZL (Sharma et al, 2021). This technique helped establishing a quantitative relationship that explains the effect of DAZL on mRNA levels and ribosome association, which appeared to correlate with the cumulative probability of DAZL binding within clusters of proximal 3' UTR binding sites.…”

A specialized mRNA translation circuit instated in pluripotency presets the competence for cardiogenesis in humans

“…Moreover the composition of the RNA interactomes of RBPs is context-dependent and dynamic (Sharma et al, 2021). Therefore, we reasoned that association of specific RBPs with ribosomal complexes could specialize their translational output by selectively translating specific groups of mRNAs.…”