Interactions between RNA-binding proteins (RBPs) and RNAs are critical to cell biology. However, methods to comprehensively and quantitatively assess these interactions within cells were lacking. RNA interactome capture (RIC) uses in vivo UV crosslinking, oligo(dT) capture, and proteomics to identify RNA-binding proteomes. Recent advances have empowered RIC to quantify RBP responses to biological cues such as metabolic imbalance or virus infection. Enhanced RIC exploits the stronger binding of locked nucleic acid (LNA)-containing oligo(dT) probes to poly(A) tails to maximize RNA capture selectivity and efficiency, profoundly improving signal-to-noise ratios. The subsequent analytical use of SILAC and TMT proteomic approaches, together with high-sensitivity sample preparation and tailored statistical data analysis, substantially improves RIC’s quantitative accuracy and reproducibility. This optimized approach is an extension of the original RIC protocol. It takes 3 d plus 2 weeks for proteomics and data analysis and will enable the study of RBP dynamics under different physiological and pathological conditions. This protocol extension describes an improved method for global profiling of poly(A) RNA-binding proteins (RBPs) and quantitative analysis of RBP dynamics in response to biological and pharmacological cues that uses UV crosslinking, capture with LNA-modified oligo-dT probes, and proteomics.

/pdf/global-analysis-of-rna-binding-protein-dynamics-by-569zjv6tls.pdf

Global analysis of RNA-binding protein dynamics by comparative and enhanced RNA interactome capture.

Hundreds of canonical RNA binding proteins facilitate diverse and essential RNA processing steps in cells forming a central regulatory point in gene expression. However, recent discoveries including the identification of a large number of noncanonical proteins bound to RNA have changed our view on RNA-protein interactions merely as necessary steps in RNA biogenesis. As the list of proteins interacting with RNA has expanded, so has the scope of regulation through RNA-protein interactions. In addition to facilitating RNA metabolism, RNA binding proteins help to form subcellular structures and membraneless organelles, and provide means to recruit components of macromolecular complexes to their sites of action. Moreover, RNA-protein interactions are not static in cells but the ribonucleoprotein (RNP) complexes are highly dynamic in response to cellular cues. The identification of novel proteins in complex with RNA and ways cells use these interactions to control cellular functions continues to broaden the scope of RNA regulation in cells and the current challenge is to move from cataloguing the components of RNPs into assigning them functions. This will not only facilitate our understanding of cellular homeostasis but may bring in key insights into human disease conditions where RNP components play a central role. This review brings together the classical view of regulation accomplished through RNA-protein interactions with the novel insights gained from the identification of RNA binding interactomes. We discuss the challenges in combining molecular mechanism with cellular functions on the journey towards a comprehensive understanding of the regulatory functions of RNA-protein interactions in cells. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications aRNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition.

From parts lists to functional significance-RNA-protein interactions in gene regulation.


 Plants selectively transport mobile mRNAs through intercellular pores, plasmodesmata (PD), to distribute spatial information for synchronizing meristematic differentiation with environmental dynamics. However, how plants recognize and deliver mobile mRNAs to PD remains unknown. Here, by using RNA-live cell imaging, we show that mobile mRNAs hitchhike on organelle trafficking to transport to PD. Perturbed cytoskeleton organization or organelle trafficking severely disrupts the subcellular distribution of mobile mRNAs. We further show that Arabidopsis rotamase cyclophilins (ROCs), which are organelle-localized RNA-binding proteins (RBPs), specifically bind mobile mRNAs on the surface of organelles to direct PD-targeting. Arabidopsis roc quadruple mutants showed reduced in PD-targeting of mobile mRNAs, along with phenotype alterations. ROCs can move intercellularly and form RNA-protein complexes in phloem, suggesting the roles of ROCs in delivery of mobile mRNAs through PD. Our results highlight that an RBP-mediated hitchhiking system is purposely recruited to orient plant-mobile mRNAs to PD for intercellular transport.

Arabidopsis cyclophilins direct plasmodesmata-targeting of mobile mRNA via organelle hitchhiking

RNA-binding proteins (RBPs) play critical roles in post-transcriptional gene regulation and are known to contribute to plant immunity. To understand the responses of cellular RBPs to an immune elicitor, we applied RNA interactome capture to Arabidopsis leaves treated with flg22. Strikingly, flg22 induced a pervasive remodelling of the cellular RBPome affecting 186 proteins. Flg22-responsive RBPs included classical RBPs involved in RNA metabolism as well as non-canonical RBPs. RBP responders detected after 2h of treatment are enriched in putative sites for post-translational modifications, which may play a regulatory role. By contrast, changes in RBP abundance becomes increasingly important for the RBPome responses to flg22 after 12h. Plant resistance to Pseudomonas syringae is strongly altered in mutant lines lacking individual flg22-responsive RBPs, supporting the importance of RBP dynamics in plant immunity. This study provides a comprehensive and systematic census of flg22 responsive plant RBPs, discovering novel components of plant immunity.

/pdf/proteome-wide-profiling-of-rna-binding-protein-responses-to-41zcgil7qz.pdf

Proteome-wide Profiling of RNA-Binding Protein Responses to flg22 Reveals Novel Components of Plant Immunity

The nuclear RNA exosome plays a key role in controlling the levels of multiple protein-coding and non-coding RNAs. Recruitment of the exosome to specific RNA substrates is mediated by RNA-binding co-factors. The transient interaction between co-factors and the exosome as well as the rapid decay of RNA substrates make identification of exosome co-factors challenging. Here, we use comparative poly(A)+ RNA interactome capture in fission yeast expressing three different mutants of the exosome to identify proteins that interact with poly(A)+ RNA in an exosome-dependent manner. Our analyses identify multiple RNA-binding proteins whose association with RNA is altered in exosome mutants, including the zinc-finger protein Mub1. Mub1 is required to maintain the levels of a subset of exosome RNA substrates including mRNAs encoding for stress-responsive proteins. Removal of the zinc-finger domain leads to loss of RNA suppression under non-stressed conditions, altered expression of heat shock genes in response to stress, and reduced growth at elevated temperature. These findings highlight the importance of exosome-dependent mRNA degradation in buffering gene expression networks to mediate cellular adaptation to stress.

https://www.life-science-alliance.org/content/lsa/5/2/e202101111.full.pdf

RNA-binding protein Mub1 and the nuclear RNA exosome act to fine-tune environmental stress response.

Large RNA-binding complexes play a central role in gene expression and orchestrate production, function, and turnover of mRNAs The accuracy and dynamics of RNA-protein interactions within these molecular machines are essential for their function and are mediated by RNA-binding proteins (RBPs) Here, we show that fission yeast whole-cell poly(A)+ RNA-protein crosslinking data provide information on the organization of RNA-protein complexes To evaluate the relative enrichment of cellular RBPs on poly(A)+ RNA, we combine poly(A)+ RNA interactome capture with a whole-cell extract normalization procedure This approach yields estimates of in vivo RNA-binding activities that identify subunits within multiprotein complexes that directly contact RNA As validation, we trace RNA interactions of different functional modules of the 3' end processing machinery and reveal additional contacts Extending our analysis to different mutants of the RNA exosome complex, we explore how substrate channeling through the complex is affected by mutation Our data highlight the central role of the RNA helicase Mtl1 in regulation of the complex and provide insights into how different components contribute to engagement of the complex with substrate RNA In addition, we characterize RNA-binding activities of novel RBPs that have been recurrently detected in the RNA interactomes of multiple species We find that many of these, including cyclophilins and thioredoxins, are substoichiometric RNA interactors in vivo Because RBPomes show very good overall agreement between species, we propose that the RNA-binding characteristics we observe in fission yeast are likely to apply to related proteins in higher eukaryotes as well

/pdf/system-wide-analyses-of-the-fission-yeast-poly-a-rna-chsacnwd5k.pdf

System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organization and function of RNA-protein complexes.

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System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organization and function of RNA-protein complexes.