CRISPR screen to identify lncRNAs affecting protein stability

Long non-coding RNAs (lncRNAs) defined as transcripts longer than 200 nucleotides, they are not translated into protein. The function of most lncRNAs are not known yet, but over the last decade researches have begun to emphasize the importance of the function of many lncRNAs, linking them to many diseases and biological functions, including transcriptional regulation, post transcriptional regulation and other functions.

We decided to study the role of lncRNAs in post transcriptional regulation, focusing on the role of lncRNAs in the regulation of degron levels. Degron is a short amino acid sequence which is part of the protein that play role in regulation protein degradation, this sequence is recognized by E3 ubiquitin ligases and target proteins for degradation.

In my study we focus on the importance of degrons in two different oncogenes and one tumor-suppressor: b-catenin (CTNNB1), n-Myc (MYCN) and p14ARF(CDKN2A). To identify lncRNAs regulating degron levels, we performed a CRISPR interference (CRISPRi) screen and measured the protein stability using Global Protein Stability system (GPS). GPS system uses two florescent proteins as a reporter: DsRed and EGFP and between them IRES. IRES is an RNA element that allows translation initiation in a cap independent manner, EGFP and DsRed proteins are derived from the same mRNA but each one expressed separately

Our aim is to find functional lncRNA genes affecting protein stability followed by validation of the enriched genes. Identification of those lncRNAs will help us understand the mechanism of protein degradation and could potentially reveal novel therapeutic targets in cancer