Details

Autor(en) / Beteiligte

• Shchepachev, Vadim
• Bresson, Stefan
• Spanos, Christos
• Petfalski, Elisabeth
• Fischer, Lutz
• Rappsilber, Juri
• Tollervey, David

Titel

Defining the RNA interactome by total RNA‐associated protein purification

Ist Teil von

• Molecular systems biology, 2019-04, Vol.15 (4), p.e8689-n/a

Ort / Verlag

England: John Wiley & Sons, Inc

Erscheinungsjahr

2019

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• 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 hundreds of RNA binding proteins, strongly enriched for canonical RBPs. 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 tRNAs. 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 RBPome characterization. Synopsis This study presents TRAPP (total RNA‐associated protein purification), a large‐scale approach that allows the rapid identification of all RNA‐binding proteins and quantification of dynamic changes following exposure to stress. TRAPP allows rapid identification of the RNA‐bound proteome in both eukaryotes (S. cerevisiae) and bacteria (E. coli). PAR‐TRAPP in yeast quantified changes in the RNA‐bound proteome on stress, revealing specific defects in 60S ribosome maturation. iTRAPP mapped 524 unique RNA‐peptide crosslinks from 178 proteins, with amino acid resolution. This study presents TRAPP (total RNA‐associated protein purification), a large‐scale approach that allows the rapid identification of all RNA‐binding proteins and quantification of dynamic changes following exposure to stress.