Quantitative Profiling of In Vivo-assembled RNA-Protein Complexes Using a Novel Integrated Proteomic Approach

Abstract

-1, eIF-2A, and eIF3g), and ribosomal subunits with known noncanonical actions (RPS19, RPS7, and RPL26). Validation studies with the small molecule eIF4A-1 inhibitor Hippuristanol shows that translation of endogenous lymphoid enhancer factor-1 mRNA is especially sensitive to eIF4A-1 activity. Our work demonstrates that MS2 in vivo biotin tagged RNA affinity purification is an effective and versatile approach

Investigation of protein–RNA interactions by mass spectrometry—Techniques and applications

“…Another recently published strategy is the MS2-BioTRAP. This involves affinity purification of biotin-tagged MS2 loops of RNA, and it thus allows the isolation of protein-RNA complexes assembled in vivo through their RNA moiety [29].…”

“…Background protein-binding showed equal abundance in both samples, whereas specifically binding proteins showed high enrichment in the pull-down experiments using the bait RNA [54]. A similar strategy that implements SILAC has been introduced by Tsai et al [29]. They used MS2-BioTRAP (see above) to identify and quantify LEF1 IRES RNA-binding proteins.…”

Translational Control in Cancer Etiology

“…The combination of these two technologies may provide a very accurate portrait of how mRNA secondary structures control the translation of the cancer genome. Furthermore, techniques such as high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) and affinity RNA purification followed by mass spectrometry have successfully identified cis-regulatory elements and specific regulatory factors associated with RNAs (Ji et al 2004;Darnell et al 2011;Tsai et al 2011;Darnell and Richter 2012). Such technologies could be used to characterize the mRNAs and the translational complex directly bound to eIFs such as eIF3, eIF4G, eIF5B, and eIF4A.…”

Quantitative interaction screen of telomeric repeat-containing RNA reveals novel TERRA regulators

“…SILAC determines the intensity of specific ''light'' and ''heavy'' peptide pairs that are mass shifted from each other due to metabolic labeling of the proteome with isotopeenriched amino acids (Mann 2006). So far, SILAC-based quantitative mass spectrometry for the identification of RNA-associated proteins has been used to investigate selected mRNA fragments (Butter et al 2009;Tsai et al 2011;Ward et al 2011;Scheibe et al 2012) or polyadenylated RNA on a global scale (Baltz et al 2012). For the first time, we here apply quantitative interaction proteomics to systematically and comprehensively determine proteins interacting with a noncoding repetitive RNA, leading to the identification of novel interactors with a role in TERRA homeostasis and its association with telomeres.…”