Identification of Candidate RNA Binding Proteins Associated with RNPS1 3’UTR

Abstract

The course of the expedition of RNA–from RNA biosynthesis to RNA degradation—is determined by a wide number of proteins that associate with RNA to moderate its fate. The interaction between RNA and protein can be mediated through RNA-binding proteins (RBPs) or auxiliary proteins, which act cooperatively within multisubunit ribonucleoprotein (RNP) complexes. As a consequence, identifying the RBP-RNA interaction is essential in comprehending how genes are regulated. The commonly used strategy is to isolate RNAs bound to known RBPs; however, recently, pull-down methods have been developed for the direct precipitation of specific RNA of interest and its associated proteins. We have shown that RNPS1 is subjected to post-transcriptional gene regulation via miRNAs. Herein, we performed a systematic method for verifying the regulatory RBPs linked with RNPS1 3’UTR in the physiological conditions. The method employs the RNA sequence of MS2 followed by affinity purification. The RNPS1 3’UTR is labeled with MS2 RNA hairpins and the chimeric RNA is then precipitated-down by the cognate expression of Flag-MS2 coat protein. Consequent assessment of the eluted ribonucleoprotein (RNP) complex through mass-spectrometry determined a list of proteins, which interact with RNPS1 3’UTR. These proteins plausibly govern the fate of RNPS1 mRNA, including transcription, splicing, localization, translation and decay.