Defining the RNA Interactome by Total RNA-Associated Protein Purification

Abstract

UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. We sought to develop a technique to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, total RNA-associated protein purification (TRAPP), was applied to yeast (S. cerevisiae) and bacteria (E. coli). In all analyses, SILAC labelling was used to quantify protein recovery in the presence and absence of irradiation. For S. cerevisiae, we also compared crosslinking using 254 nm (UVC) irradiation (TRAPP) with 4-thiouracil (4tU) labelling combined with ~350 nm (UVA) irradiation (PAR-TRAPP). Recovery of proteins not anticipated to show RNA-binding activity was substantially higher in TRAPP compared to PAR-TRAPP. As an example of preferential TRAPP-crosslinking, we tested enolase (Eno1) and demonstrated its binding to tRNA loops in vivo. We speculate that many protein-RNA interactions have biophysical effects on localization and/or accessibility, by opposing or promoting phase separation for highly abundant protein. Homologous metabolic enzymes showed RNA crosslinking in S. cerevisiae and E. coli, indicating conservation of this property. TRAPP allows alterations in RNA interactions to be followed and we initially analyzed the effects of weak acid stress. This revealed specific alterations in RNA-protein interactions; for example, during late 60S ribosome subunit maturation. Precise sites of crosslinking at the level of individual amino acids (iTRAPP) were identified in 395 peptides from 155 unique proteins, following phospho-peptide enrichment combined with a bioinformatics pipeline (Xi). TRAPP is quick, simple and scalable, allowing rapid characterization of the RNA-bound proteome in many systems.

Actin remodelling controls proteasome homeostasis upon stress

“…3c ). This is consistent with a recent study that identified Ede1 as a potential RNA-binding protein 22 . To confirm that Ede1 is regulating ADC17 mRNA at the level of translation, we deployed the SunTag labelling method 23 .…”

Systematic mapping of protein‐metabolite interactions in central metabolism of Escherichia coli

“…While ATP was known to bind to a variety of proteins in several organisms (Reinhard et al , ; Geer & Fitzgerald, ; Piazza et al , ), promiscuous binding of GTP was reported only recently (Piazza et al , ). The frequent binding of purine nucleotides does not appear to be explained by propensity of specific proteins for RNA binding, since we found no correlation with RNA binding enzymes from two recently published E. coli studies (Queiroz et al , ; Shchepachev et al , ). The recent discovery of purine nucleotide function as biological hydrotropes (Patel et al , ) could offer an explanation; however, effects on protein aggregation were only observed at much higher concentrations than used in our assay (> 5 mM compared to 0.2 mM).…”

Single Nucleotide Resolution RNA–Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow

“…Different approaches are available to study RNA–protein interactions from a protein-centric view (to identify novel RNA-binding proteins and domains) or from an RNA-centric view (to identify novel RNA targets of specific RNA-binding proteins (RBP)). The latter group of methods, e.g., CLIP-Seq (cross-linking immunoprecipitation-high-throughput sequencing), involves next-generation sequencing after affinity purification of an RBP cross-linked to its target RNAs. − In contrast, protein-centric analyses identify proteins bound to a specific RNA or a subset of RNAs employing, e.g., cross-linking by UV light and mass spectrometry. ,− Such a method was used to characterize the mRBPome by cross-linking mRNA-bound proteins and subsequent purification of mRNAs by oligo-dT chromatography . The specific amino acid residue that cross-linked to an RNA is identified using mass spectrometry .…”