Identification and initial characterization of POLIII-driven transcripts by msRNA-sequencing

Non-coding RNAs (ncRNAs) are powerful regulators of gene expression but medium-sized (50-300 nts in length) ncRNAs (msRNAs) are barely picked-up precisely by RNA-sequencing. Here we describe msRNA-sequencing (msRNAseq), a modified protocol that associated with a computational analyses pipeline identified about similar to 1800 msRNA loci, including over 300 putatively novel msRNAs, in human and murine cells. We focused on the identification and initial characterization of three POLIII-derived transcripts. The validation of these uncharacterized msRNAs identified an ncRNA in antisense orientation from the POLR3E locus transcribed by POLIII. This msRNA, termed POLAR (POLR3E Antisense RNA), has a strikingly short half-life, localizes to paraspeckles (PSPs) and associates with PSP-associated proteins indicating that msRNAseq identifies functional msRNAs. Thus, our analyses will pave the way for analysing the roles of msRNAs in cells, development and diseases.