Comprehensive in vivo identification of the c-Myc mRNA protein interactome using HyPR-MS

Abstract

Proteins bind mRNA through their entire life cycle from transcription to degradation. We analyzed c-Myc mRNA protein interactors in vivo using the HyPR-MS method to capture the crosslinked mRNA by hybridization and then analyzed the bound proteins using mass spectrometry proteomics. Using HyPR-MS, 229 c-Myc mRNA-binding proteins were identified, confirming previously proposed interactors, suggesting new interactors, and providing information related to the roles and pathways known to involve c-Myc. We performed structural and functional analysis of these proteins and validated our findings with a combination of RIP-qPCR experiments, in vitro results released in past studies, publicly available RIP-and eCLIP-seq data, and results from software tools for predicting RNA-protein interactions.

Chaperonin-Containing TCP1 Complex (CCT) Promotes Breast Cancer Growth Through Correlations With Key Cell Cycle Regulators

“…Our data also suggests that additional mechanisms beyond protein-folding and biogenesis may directly influence the gene expression of key cell cycle regulators like CCND1 and CDK2 and growth promoters like MYC. One way could be by acting as an RNA binding protein (RBP), since CCT2 was shown to interact with MYC mRNA and could govern the biology of this mRNA (60). How CCT2 increases the expression of CCND1 or CDK2 remains unknown but could be an indirect result of increasing MYC or other transcription factors.…”

Identifying HIV-1 RNA splice variant protein interactomes using HyPR-MSSV

“…Purification of HIV-1 splice variant classes. We recently described HyPR-MS (Hybridization Purification of RNA-Protein Complexes Followed by Mass Spectrometry); a strategy to identify the in vivo protein interactomes of specific viral RNAs, lncRNAs, and mRNAs (Knoener et al, 2017;Spiniello et al, 2018;Spiniello et al, 2019). Here, we present HyPR-MSsv, a strategy that expands the capabilities of HyPR-MS to differentiate in vivo protein interactomes for multiple splice variants (SV) derived from a single primary transcript and isolated from a single cell population.…”

RNA-Centric Approaches to Profile the RNA–Protein Interaction Landscape on Selected RNAs

“…In total, 229 proteins were found to be associated with c-myc compared to control samples (≥5-fold), including several RBPs previously known to interact with the mRNA. It also revealed novel interactors and comparison with published CLIP/RIP data strongly suggested those being bona fide interactors [71]. Conversely, vIPR (in vivo Interactions by Pull-down of RNA) used a tiling approach, using multiple 3 -biotinylated TEG linked 20-mer oligonucleotides to recover GFP-tagged gld-1 mRNA from the nematode Caenorhabditis elegans [77].…”

“…Two recent studies have successfully implemented short unmodified DNA oligonucleotides to capture mRNAs from cells. Applying HyPR-MS, c-myc mRNA, coding for one of the best studied oncogenes, was captured from formaldehyde crosslinked K562 cells [ 71 ]. Therefore, two DNA oligonucleotides were used to ensure uniform capture of the full c-myc transcript, while a scrambled oligo was used as control.…”