RNA-Decoder : Development of a novel DNA-sequencing-based method to identify proteins binding to a single specific RNA transcript

Abstract

Long non-coding RNAs (lncRNAs) constitute a diverse class of RNA-molecules that serve as important regulators of various cellular processes such as metabolism, despite not encoding proteins. Typically, lncRNAs do not act alone within cells; instead, they interact with RNA-binding proteins to form functional ribonucleoprotein complexes (RNPs). RNPs can alter gene expression through epigenetic mechanisms by modelling chromatin structure or affecting transcription or translation. The aim of this study is to develop a new method called RNA-Decoder which focuses on identifying the RNA-binding proteins within RNPs that interact with protein coding RNA transcripts (mRNA) and lncRNA transcripts originating from a divergent transcription locus called GCG1-SUT098 in S. cerevisiae. RNA-Decoder utilizes Epi-Decoder yeast library which consists of thousands of yeast strains, each containing two unique DNA-barcodes at the locus of interest and a unique protein with a tandem immunopurification tag (TAP-tag). In RNA-Decoder method, RNPs formed in the S. cerevisiae cells were isolated by TAP-tag immunoprecipitation, and gathered RNA transcripts from isolated RNPs are transcribed into cDNA. cDNA was amplified by PCR to be able to sequence the barcoded regions. Sequencing results enable comparison between mRNA and lncRNA interacting RNA-binding proteins. The preliminary results of RNA-Decoder indicate its potentiality, and functionality. Western blot - analysis indicates successful immunoprecipitation of RNPs. The correct sizes PCR products from barcoded region of cDNAs indicate successful cDNA synthesis and barcode amplification. However, PCR reaction targeting GCG1 barcode region would require more optimization due primer-dimer formation during the reaction. To validate RNA-Decoder method and to detect the differences between mRNA-protein and lncRNA-protein complexes the amplified barcode regions needs to be sequenced in the future.