Unbiased RNA-protein interaction screen by quantitative proteomics.
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.
Butter
Falk
F
Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
Scheibe
Marion
M
Mörl
Mario
M
Mann
Matthias
M
D020413:3' Untranslated Regions / Q000235:genetics; D020121:5' Untranslated Regions / Q000235:genetics; D000596:Amino Acids / Q000378:metabolism; D000954:Antigens, Surface / Q000378:metabolism; D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D006367:HeLa Cells; D056464:Histone Deacetylase 2; D006655:Histone Deacetylases / Q000235:genetics; D006657:Histones / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011506:Proteins / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics; D015534:Trans-Activators
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000596:Amino Acids; D000954:Antigens, Surface; D052157:Aptamers, Nucleotide; C474043:DGCR8 protein, human; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D006657:Histones; C089320:MTA1 protein, human; D011506:Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D012313:RNA; D056464:Histone Deacetylase 2; D006655:Histone Deacetylases