A combined RNAi and localization approach for dissecting long noncoding RNAs reveals a function of Panct1 in ES cells

Long non-coding RNAs (lncRNAs) regulate diverse biological pathways. Unlike protein coding genes, where methods to comprehensibly study their functional roles in cellular systems are available, techniques to systematically investigate lncRNAs have largely remained unexplored. Here, we report a technology for combined Knockdown and Localization Analysis of Non-coding RNAs (c-KLAN) that merges phenotypic characterization and localization approaches to study lncRNAs. Using a library of endoribonuclease prepared short interfering RNAs (esiRNAs) coupled with a pipeline for synthesizing labeled riboprobes for RNA fluorescence in situ hybridization (FISH), we demonstrate the utility of c-KLAN by identifying a novel transcript Panct1 (Pluripotency associated non-coding transcript 1) that regulates embryonic stem cell identity. We postulate that c-KLAN should be generally useful in the discovery of lncRNAs implicated in various biological processes.

6

Chakraborty D, Kappei D, Theis M, Nitzsche A, Ding L, Paszkowski-Rogacz M, Surendranath V, Berger N, Schulz H, Saar K, Hubner N, Buchholz F