Unbiased RNA-protein interaction screen by quantitative proteomics.

Abstract:

:Mass spectrometry (MS)-based quantitative interaction proteomics has successfully elucidated specific protein-protein, DNA-protein, and small molecule-protein interactions. Here, we developed a gel-free, sensitive, and scalable technology that addresses the important area of RNA-protein interactions. Using aptamer-tagged RNA as bait, we captured RNA-interacting proteins from stable isotope labeling by amino acids in cell culture (SILAC)-labeled mammalian cell extracts and analyzed them by high-resolution, quantitative MS. Binders specific to the RNA sequence were distinguished from background by their isotope ratios between bait and control. We demonstrated the approach by retrieving known and novel interaction partners for the HuR interaction motif, H4 stem loop, "zipcode" sequence, tRNA, and a bioinformatically-predicted RNA fold in DGCR-8/Pasha mRNA. In all experiments we unambiguously identified known interaction partners by a single affinity purification step. The 5' region of the mRNA of DGCR-8/Pasha, a component of the microprocessor complex, specifically interacts with components of the translational machinery, suggesting that it contains an internal ribosome entry site.

authors

Butter F,Scheibe M,Mörl M,Mann M

doi

10.1073/pnas.0812099106

subject

Has Abstract

pub_date

2009-06-30 00:00:00

pages

10626-31

issue

26

eissn

0027-8424

issn

1091-6490

pii

0812099106

journal_volume

106

pub_type

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