RNA-protein interactomes as invaluable resources to study RNA viruses: Insights from SARS CoV-2 studies.

Understanding the molecular mechanisms of severe respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is essential for the successful development of therapeutic strategies against the COVID-19 pandemic. Numerous studies have focused on the identification of host factors and cellular pathways involved in the viral replication cycle. The speed and magnitude of hijacking the translation machinery of host mRNA, and shutting down host transcription are still not well understood. Since SARS-CoV-2 relies on host RNA-binding proteins for the infection progression, several efforts have been made to define the SARS-CoV-2 RNA-bound proteomes (RNA-protein interactomes). Methodologies that enable the systemic capture of protein interactors of given RNA in vivo have been adapted for the identification of the SARS-CoV-2 RNA interactome. The obtained proteomic data aided by genome-wide and targeted CRISPR perturbation screens, revealed host factors with either pro- or anti-viral activity and highlighted cellular processes and factors involved in host response. We focus here on the recent studies on SARS-CoV-2 RNA-protein interactomes, with regard to both the technological aspects of RNA interactome capture methods and the obtained results. We also summarize several related studies, which were used in the interpretation of the SARS-CoV-2 RNA-protein interactomes. These studies provided the selection of host factors that are potentially suitable candidates for antiviral therapy. Finally, we underscore the importance of RNA-protein interactome studies in regard to the effective development of antiviral strategies against current and future threats. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA in Disease and Development > RNA in Disease RNA Methods > RNA Analyses in Cells.

Transcriptome-wide Identification of RNA-binding Protein Binding Sites Using Photoactivatable-Ribonucleoside-Enhanced Crosslinking Immunoprecipitation (PAR-CLIP).

RNA-binding proteins (RBPs) mediate important co- and post-transcriptional gene regulation by binding pre-mRNA in a sequence- and/or structure-specific manner. For a comprehensive understanding of RBP function, transcriptome-wide mapping of the RNA-binding sites is essential, and CLIP-seq methods have been developed to elucidate protein/RNA interactions at high resolution. CLIP-seq combines protein/RNA UV-crosslinking with immunoprecipitation (CLIP) followed by high-throughput sequencing of crosslinked RNA fragments. To overcome the limitations of low RNA-protein crosslinking efficiency in standard CLIP-seq, photoactivatable-ribonucleoside-enhanced CLIP (PAR-CLIP) has been developed. Here, living cells or whole organisms are fed photo-activatable nucleoside analogs that are incorporated into nascent RNA transcripts before UV treatment. This allows greater crosslinking efficiency at comparable radiation doses for enhanced RNA recovery and separation of crosslinked target RNA fragments from background RNA degradation products. Moreover, it facilitates the generation of specific UV-induced mutations that mark the crosslinking nucleotide and allow transcriptome-wide identification of RBP binding sites at single-nucleotide resolution. © by 2017 John Wiley & Sons, Inc.

The mRNA-bound proteome of the early fly embryo.

Early embryogenesis is characterized by the maternal to zygotic transition (MZT), in which maternally deposited messenger RNAs are degraded while zygotic transcription begins. Before the MZT, post-transcriptional gene regulation by RNA-binding proteins (RBPs) is the dominant force in embryo patterning. We used two mRNA interactome capture methods to identify RBPs bound to polyadenylated transcripts within the first 2 h of Drosophila melanogaster embryogenesis. We identified a high-confidence set of 476 putative RBPs and confirmed RNA-binding activities for most of 24 tested candidates. Most proteins in the interactome are known RBPs or harbor canonical RBP features, but 99 exhibited previously uncharacterized RNA-binding activity. mRNA-bound RBPs and TFs exhibit distinct expression dynamics, in which the newly identified RBPs dominate the first 2 h of embryonic development. Integrating our resource with in situ hybridization data from existing databases showed that mRNAs encoding RBPs are enriched in posterior regions of the early embryo, suggesting their general importance in posterior patterning and germ cell maturation.