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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 labeling was used to quantify protein recovery in the presence and absence of irradiation. For S.cerevisiae, we also compared crosslinking using 254nm (UVC) irradiation (TRAPP) with 4-thiouracil (4SU) labeling combined with 350nm (UVA) irradiation (PAR-TRAPP). Recovery of proteins not anticipated to show RNA-binding activity was significantly higher in TRAPP compared to PAR-TRAPP. As an example of preferential TRAPP-crosslinking, we tested enolase (Eno1) and demonstrated its strong, but largely sequence independent, binding to RNA in vivo. We speculate that many protein-RNA interactions have biophysical effects on localization and/or accessibility, by opposing or promoting phase separation. 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 following phospho-peptide enrichment combined with a bioinformatic pipeline (Xi). TRAPP is quick, simple and scalable, allowing rapid characterization of the RNA-bound proteome in many systems.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE119867 | GEO | 2018/09/13

REPOSITORIES: GEO

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Project description:Protein-RNA interactions are integral components of nearly every aspect of biology including regulation of gene expression, assembly of cellular architectures, and pathogenesis of human diseases. However, studies in the past few decades have only uncovered a small fraction of the vast landscape of the protein-RNA interactome in any organism, and even less is known about the dynamics of protein-RNA interactions under changing developmental and environmental conditions. Here, we describe the gPAR-CLIP (global photoactivatable-ribonucleoside-enhanced crosslinking and immunopurification) approach for capturing regions of the transcriptome bound by RNA-binding proteins (RBPs) in budding yeast. We report over 13,000 RBP crosslinking sites in untranslated regions (UTR) covering 72% of protein-coding transcripts encoded in the genome, confirming 3M-bM-^@M-^Y UTRs as major sites for RBP interaction. Comparative genomic analyses reveal that RBP crosslinking sites are highly conserved, and RNA folding predictions indicate that secondary structural elements are constrained by protein binding and may serve as generalizable modes of RNA recognition. Finally, 38% of 3M-bM-^@M-^Y UTR crosslinking sites show changes in RBP occupancy upon glucose or nitrogen deprivation, with major impacts on metabolic pathways as well as mitochondrial and ribosomal gene expression. Our study offers an unprecedented view of the pervasiveness and dynamics of protein-RNA interactions in vivo. Duplicate gPAR-CLIP and mRNA-seq libraries were sequenced from yeast strains for each of three conditions: log-phase growth, growth after 2 hour glucose starvation, and growth after 2 hour nitrogen starvation. Additional duplicate mRNA-seq libraries were sequenced from yeast strains grown in the absence of 4-thiouracil. gPAR-CLIP libraries were used to determine regions of mRNA bound by proteins. mRNA-seq libraries served as controls for mRNA abundance. A Puf3p PAR-CLIP library was sequenced to determine how well gPAR-CLIP captured the binding signatures of a single RNA-binding protein.

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Defining the RNA Interactome by Total RNA-Associated Protein Purification

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