Proteomics

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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), relies on SILAC labelling to quantify RNA-associated protein recovery in the presence and absence of irradiation. We utilised TRAPP to study alterations in RNA-protein interactions upon exposure to weak acid stress in yeast. In addition, as UV irradiation induces short distance crosslinks between proteins and nucleic acids, the identity of crosslinked amino acids reveals the exact protein-RNA interacting interface. Precise sites of crosslinking at the level of individual amino acids (iTRAPP) were identified following phospho-peptide enrichment combined with a bioinformatic pipeline (Xi).

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Escherichia Coli Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Vadim Shchepachev  

LAB HEAD: David Tollervey

PROVIDER: PXD011071 | Pride | 2019-03-26

REPOSITORIES: Pride

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Defining the RNA interactome by total RNA-associated protein purification.

Shchepachev Vadim V   Bresson Stefan S   Spanos Christos C   Petfalski Elisabeth E   Fischer Lutz L   Rappsilber Juri J   Tollervey David D  

Molecular systems biology 20190408 4

The RNA binding proteome (RBPome) 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), RNAs were labelled with 4-thiouracil prior to 350 nm crosslinking. PAR-TRAPP in yeast identified hundr  ...[more]

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