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Identifying IRES RNA‐Protein Complexes by SILAC based Mass Spectrometry
Author(s) -
Tsai Becky Pin,
Wang Xiaorong,
Huang Lan,
Waterman Marian L
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.499.11
Subject(s) - internal ribosome entry site , stable isotope labeling by amino acids in cell culture , rna , translation (biology) , ribosome , biotinylation , microbiology and biotechnology , messenger rna , biology , computational biology , chemistry , proteomics , biochemistry , gene
The dynamic nature of RNA protein complexes has made it challenging to study post‐transcriptional regulation in development and disease. We have developed a strategy to rapidly isolate and characterize in vivo assembled protein complexes for specific RNAs. Our proof‐of‐principle RNA target is a 1.2 kb IRES (internal ribosome entry site) in the 5′UTR region of Lymphoid Enhancer Factor‐1 (LEF1) mRNA. IRESes provide an alternative mechanism of ribosome recruitment and translation that is not well understood. To isolate in vivo assembled IRES‐protein complexes, the LEF1 IRES is engineered with a cluster of stem‐loops recognized by in vivo biotinylated MS2 bacteriophage coat proteins. The MS2 protein is used to rapidly isolate the complex with magnetic streptavidin beads. Our strategy is designed to isolate in vivo assembled RNA‐protein complexes for LC MS/MS identification of IRES binding proteins. SILAC‐based quantitative MS is used to detect proteins enriched on the target LEF1 IRES vs. non‐IRES mRNA. Results include known and putative IRES binding factors that were moderately enriched on the LEF1 IRES. The finding suggests that IRES elements assemble complexes of canonical RNA regulatory factors, rather than a few dedicated factors. In summary, our method provides a versatile and highly adaptable approach that can be applied to different RNA‐protein interactions.