Identifying lncRNA–Protein Interactions in Hematopoietic Progenitor Cells by Hybridization Capture and Mass Spectrometry

Abstract

Long noncoding RNAs (lncRNAs) exert regulatory functions in a wide spectrum of biological contexts, and certain regulatory functions involve the formation of RNA–protein complexes. Discovering the structure and function of these complexes may unveil important functional insights. The DDX41 gene encoding the DEAD-box RNA helicase 41 protein (DDX41) is subject to extensive germline genetic variation, and certain variants create a predisposition to develop myelodysplastic syndrome and acute myeloid leukemia. While the importance of DDX41 for the control of hematopoiesis is established, many questions remain regarding the mechanisms of how DDX41 functions in hematopoietic stem and progenitor cells. Previously, we identified a DDX41-regulated lncRNA, growth-arrest-specific 5 (Gas5). As the Gas5 function in hematopoiesis is unknown, we analyzed the protein interactors of Gas5 lncRNA using HyPR-MS (hybridization purification of RNA–protein complexes, followed by mass spectrometry). A total of 303 proteins were identified as Gas5 lncRNA interactors, five of which were experimentally validated as Gas5 lncRNA interactors by RNA immunoprecipitation qPCR (RIP-qPCR) analysis. The identification of protein interactors with a DDX41-regulated lncRNA establishes a foundation on which to guide future mechanistic and biological studies.