RESUMO
BACKGROUND: The 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. RESULTS: We 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. CONCLUSIONS: 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.
Assuntos
RNA Longo não Codificante/genética , Núcleo Celular , Cromatina/genética , HumanosRESUMO
Single-strand breaks (SSBs) represent the major form of DNA damage, yet techniques to map these lesions genome-wide with nucleotide-level precision are limited. Here, we present a method, termed SSiNGLe, and demonstrate its utility to explore the distribution and dynamic changes in genome-wide SSBs in response to different biological and environmental stimuli. We validate SSiNGLe using two very distinct sequencing techniques and apply it to derive global profiles of SSBs in different biological states. Strikingly, we show that patterns of SSBs in the genome are non-random, specific to different biological states, enriched in regulatory elements, exons, introns, specific types of repeats and exhibit differential preference for the template strand between exons and introns. Furthermore, we show that breaks likely contribute to naturally occurring sequence variants. Finally, we demonstrate strong links between SSB patterns and age. Overall, SSiNGLe provides access to unexplored realms of cellular biology, not obtainable with current approaches.
Assuntos
Quebras de DNA de Cadeia Simples , DNA de Cadeia Simples/genética , Genoma Humano/genética , Genômica/métodos , Nucleotídeos/genética , Animais , Senescência Celular/genética , Éxons/genética , Células HeLa , Humanos , Íntrons/genética , Células K562 , Camundongos , Nucleotídeos/isolamento & purificação , SoftwareRESUMO
A convenient "one-pot" base-promoted synthesis of polysubstituted pyridines from 1-arylethylamines and ynones through the direct ß-C(sp(3))-H functionalization of enaminones under metal-free conditions has been developed. An intermolecular Michael addition reaction and an intramolecular condensation were involved in this procedure, which features high regioselectivity, high efficiency, and environmental friendliness. Various polysubstituted pyridines were provided in up to 92% yield for 34 examples.