Abstract
BackgroundThe majority of the human genome is transcribed in the form of long non-coding (lnc) RNAs. While these transcripts have attracted considerable interest, their molecular mechanisms of function and biological significance remain controversial. One of the main reasons behind this lies in the significant challenges posed by lncRNAs requiring the development of novel methods and concepts to unravel their functionality. Existing methods often lack cross-validation and independent confirmation by different methodologies and therefore leave significant ambiguity as to the authenticity of the outcomes. Nonetheless, despite all the caveats, it appears that lncRNAs may function, at least in part, by regulating other genes via chromatin interactions. Therefore, the function of a lncRNA could be inferred from the function of genes it regulates. In this work, we present a genome-wide functional annotation strategy for lncRNAs based on identification of their regulatory networks via the 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.ResultsWe applied the strategy to annotate 407 very long intergenic non-coding (vlinc) RNAs belonging to a novel widespread subclass of lncRNAs. We show that vlincRNAs indeed appear to regulate multiple genes encoding proteins predominantly involved in RNA- and development-related functions, cell cycle, and cellular adhesion via a mechanism involving proximity between vlincRNAs and their targets in the nucleus. A typical vlincRNAs can be both a positive and negative regulator and regulate multiple genes both in trans and cis. Finally, we show vlincRNAs and their regulatory networks potentially represent novel components of DNA damage response and are functionally important for the ability of cancer cells to survive genotoxic stress.ConclusionsThis study provides strong evidence for the regulatory role of the vlincRNA class of lncRNAs and a potentially important role played by these transcripts in the hidden layer of RNA-based regulation in complex biological systems.
Highlights
The majority of the human genome is transcribed in the form of long non-coding RNAs
This study provides strong evidence for the regulatory role of the vlincRNA class of lncRNAs and a potentially important role played by these transcripts in the hidden layer of RNA-based regulation in complex biological systems
We assumed that a co-expression analysis based on RNA levels measured shortly after the system is perturbed and forced to adapt by altering levels of various transcripts would more likely capture direct regulatory interactions as opposed to longer time treatments that could be diluted with indirect effects
Summary
The majority of the human genome is transcribed in the form of long non-coding (lnc) RNAs. These transcripts have been implicated in virtually all major biological processes, including normal development and disease [4,5,6,7,8,9], and are hypothesized to represent a hidden layer of regulation in complex biological organisms [10, 11] Still, relevance of this class of transcripts for most part remains controversial [12], with one of the main reasons behind it being lack of understanding of molecular mechanisms of function of these RNA species [13].
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