Defining the RNA interactome by total RNA ‐associated protein purification

Abstract

The RNA binding proteome ( RBP ome) was previously investigated using UV crosslinking and purification of poly(A)‐associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA ‐associated protein purification ( TRAPP ) based on 254 nm UV crosslinking and purification of all RNA –protein complexes using silica beads. In a variant approach ( PAR ‐ TRAPP ), RNA s were labelled with 4‐thiouracil prior to 350 nm crosslinking. PAR ‐ TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBP s. In comparison, TRAPP identified many more proteins not expected to bind RNA , and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNA s. Exploiting PAR ‐ TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA –peptide conjugates, using iTRAPP , providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBP ome characterization.

RNA promotes phase separation of glycolysis enzymes into yeast G bodies in hypoxia

“…Specifically, there were 439 direct mRNA substrates of Pfk2 with 559 discrete binding sites, 1,001 mRNA substrates with 1,432 binding sites for Eno1, and 721 mRNA substrates with 1,014 binding sites for Fba1 ( Fig S1B, right panels). The results of Eno1 PAR-CLIP-seq were in agreement with a recent analysis of Eno1 in normoxic conditions by CRAC (a method that UV crosslinks and affinity purifies protein-RNA complexes under denaturing conditions (Shchepachev et al, 2019)), providing additional validation of our identified binding sites. We identified 69 out of the top 100 bound mRNAs in the CRAC dataset.…”

Fast and unbiased purification of RNA-protein complexes after UV cross-linking

“…In earlier work, we and others found that a surprisingly large number of enzymes of intermediary metabolism bind to RNA in vivo, among them the majority of enzymes in glycolysis [28,37]. For enolase (ENO-1), theses findings were confirmed and target RNAs were identified recently [38]. Here, we show that using PTex, it is likewise possible to extract RNA-binding enzymes such as ENO-1 (Fig.…”

“…Along the same lines: the landscape of bacterial RBPs has not been determined at the same depth as for eukaryotic species; a major issue being the lack of poly(A) RNA necessary for interactome capture [5]. PTex allowed us for the first time to unbiasedly screen for proteins cross-linked to RNA in Salmonella Typhimurium [12] while RBPs in E. coli were purified by OOPS [40] and TRAPP [38].…”

“…Additionally, these protocols classically use energies between 0.2 and 0.4 J/cm 2 254 nm while the studies using a laser applied 1 J 254 nm or more to a small area [43,44]. We and others have used higher UV intensities for RBP discovery as well [12,38]. However, while we are not aware of a systematic comparison of the UV bulbs we used and UV lasers, Budowsky et al [21] noted that laser-irradiation results in an additional excitation state H T which cannot be reached when using low energy sources (see Fig.…”

mSphere of Influence: Modifying an Old Method To Study RNA-Protein Interactions

“…It is worth mentioning that since coming across this paper, alternative variants of this method have been published (3–5). It turns out that three of these groups (1, 3, 4) were independently working on the same idea and coordinated efforts to simultaneously submit to bioRxiv.…”