HyPR-MS for Multiplexed Discovery of MALAT1, NEAT1, and NORAD lncRNA Protein Interactomes

Abstract

RNA-protein interactions are integral to the regulation of gene expression. RNAs have diverse functions and the protein interactomes of individual RNAs vary temporally, spatially, and with physiological context. These factors make the global acquisition of individual RNA-protein interactomes an essential endeavor. Although techniques have been reported for discovery of the protein interactomes of specific RNAs they are largely laborious, costly, and accomplished singly in individual experiments. We developed HyPR-MS for the discovery and analysis of the protein interactomes of multiple RNAs in a single experiment while also reducing design time and improving efficiencies. Presented here is the application of HyPR-MS to simultaneously and selectively isolate the interactomes of lncRNAs MALAT1, NEAT1, and NORAD. Our analysis features the proteins that potentially contribute to both known and previously undiscovered roles of each lncRNA. This platform provides a powerful new multiplexing tool for the efficient and cost-effective elucidation of specific RNA-protein interactomes.

Retracted: Long noncoding RNA MALAT1 promotes the stemness of esophageal squamous cell carcinoma by enhancing YAP transcriptional activity

“…For example, the region of 1408-1867 nt of lncRNA uc.34 binds to the 592-759 aa region of CUL4A and inhibits CUL4A nuclear export, and this process hinders the CUL4A-mediated ubiquitination of LATS1 in hepatocellular carcinoma [30]. Spiniello et al established a HyPR-MS platform and efficiently elucidated the interactomes of lncRNAs MALAT1, NEAT1, and NORAD [35]. Additionally, lncRNA THOR, a conserved cancer/ testis lncRNA, interacts with IGF2BP1 and contributes to its mRNA stabilization activities [36].…”

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

“…We have developed HyPR-MS (hybridization purification of RNA-protein complexes followed by mass spectrometry) to address these issues by simplifying the capture oligonucleotide (CO) design, decreasing the degree of crosslinking and solubilization, and developing a multiplexed release strategy to allow the study of several RNA targets simultaneously. HyPR-MS has already been used to study the proteomes of unspliced full-length HIV RNA and MALAT1-001, NEAT1, and NORAD lncRNAs (Knoener et al 2017;Spiniello et al 2018). However, these applications targeted relatively abundant transcripts (>300 copies per cell) (Spiniello et al 2018), while most RNAs have less than 50 copies per cell (Hanley et al 2013).…”

Quantitative Proteomics to Identify Nuclear RNA-Binding Proteins of Malat1

“…In addition to RNA pulldown approaches, oligonucleotide capture experiments have been conducted to obtain RBP interactomes for the complete full-length lncRNA: HyPR-MS for human MALAT1 [23] and ChIRP-MS for murine Malat1 [24]. Our overlap to both experiments is not very high.…”

“…A recent SILAC-based RNA interaction screen reported 127 enriched proteins using the 6918-8441 nt fragment of human MALAT1 and unveiled that interaction of DBC1 with this lncRNA does sequester DBC1 from SIRT1 and thereby influences p53 acetylation [22]. Furthermore, human MALAT1 was one target in the recently described multiplexed hybridization purification of RNA-protein complexes for mass spectrometry (HyPR-MS) approach that identified 127 interaction partners to the full-length human transcript [23]. Using ChIRP-MS, another group identified 23 interactors of murine Malat1, including the TEAD proteins whose interaction with Malat1 inhibits their transcriptional activity [24].…”