Non-coding RNA profiling by high throughput sequencing
Majority of the human genome is transcribed in a form of long non-coding (lnc) RNAs. While these transcripts attracted considerable interest, their molecular mechanisms of function and biological significance remain controversial. Here, we present a genome-wide functional annotation strategy for lncRNAs based on integration of three distinct types of approaches: co-expression analysis, mapping of lncRNA-chromatin interactions and assaying molecular effects of lncRNA knockdowns obtained using an inducible and highly specific CRISPR/Cas13 system. We apply the strategy to annotate 407 vlincRNAs belonging to a novel widespread subclass of lncRNAs. We show that vlincRNAs appear to regulate, positively and negatively and both in trans and cis, multiple genes encoding proteins predominantly involved in RNA- and development-related functions, cell cycle and cellular adhesion via a potential mechanism involving proximity in nucleus. Finally, we show vlincRNAs and their regulatory networks potentially represent novel components of DNA damage response.
Regulatory networks for vlincRNAs were established based on co-expression analysis using 64 RNA-seq samples obtained using single-molecule sequencing (SMS, Helicos/SeqLL platform) from total RNA isolated from K562 cells perturbed with various drug treatments.