Study of the Long Non-Coding RNA C/EBPβ-AS in Cutaneous Melanoma

Cutaneous malignant melanoma (CMM) is the ninth most common cancer type in more developed regions. Despite comprehending less than 5% of all skin cancer cases, CMM stands as the most lethal skin neoplasm, with a detectable increase in incidence throughout recent decades. While the B-rapidly accelerated fibrosarcoma (BRAF) inhibitor vemurafenib appears to be effective in ~80% of CMM patients carrying the BRAFV600E mutation, the vast majority of patients becomes resistant to treatment. Given that, it is imperative to seek new therapeutic strategies. Long non-coding RNAs (lncRNAs) are a functionally diverse class of transcripts that lack an evident protein-coding function and have over 200 nucleotides of length. The advent of growing sensitivity of RNA sequencing methods, as well as computational prediction techniques is enabling the increasing identification of such RNA transcripts. Among the few that have been functionally characterized, several have been linked to numerous aspects of carcinogenesis, with an evident role in gene expression regulation. CCAAT/Enhancer-Binding Protein β (C/EBPβ) is a transcription factor implicated in many fundamental cellular processes, including cellular senescence and proliferation. CCAAT/Enhancer-Binding Protein β Antisense (C/EBPβ-AS) is an antisense lncRNA transcribed from the reverse strand of C/EBPβ, with a genomic 5’ overlap with C/EBPβ gene, which has not previously been studied. Here we characterize biologically relevant features of C/EBPβ-AS and propose a role for C/EBPβ-AS in epigenetic regulation of C/EBPβ expression in melanoma cell lines. Moreover, we show that modulation of C/EBPβ-AS expression resensitizes vemurafenib-resistant melanoma cells to vemurafenib. Finally, we investigate the impact of modulation of C/EBPβ-AS expression in MAPK/ERK and PI3K/AKT pathways, both commonly found to be dysregulated in CMM. Taken together, our research provides new insights on an antisense lncRNA-mediated mechanism of gene regulation, with implications on CMM targeted-therapy resistance

RAD51-dependent recruitment of TERRA lncRNA to telomeres through R-loops

Telomeres- repeated, noncoding nucleotide motifs and associated proteins that are found at the ends of eukaryotic chromosomes—mediate genome stability and determine cellular lifespan. Telomeric-repeat-containing RNA (TERRA) is a class of long noncoding RNAs (lncRNAs) that are transcribed from chromosome ends; these RNAs in turn regulate telomeric chromatin structure and telomere maintenance through the telomere-extending enzyme telomerase and homology-directed DNA repair. The mechanisms by which TERRA is recruited to chromosome ends remain poorly defined. Here we develop a reporter system with which to dissect the underlying mechanisms, and show that the UUAGGG repeats of TERRA are both necessary and sufficient to target TERRA to chromosome ends. TERRA preferentially associates with short telomeres through the formation of telomeric DNA–RNA hybrid (R-loop) structures that can form in trans. Telomere association and R-loop formation trigger telomere fragility and are promoted by the recombinase RAD51 and its interacting partner BRCA2, but counteracted by the RNA-surveillance factors RNaseH1 and TRF1. RAD51 physically interacts with TERRA and catalyses R-loop formation with TERRA in vitro, suggesting a direct involvement of this DNA recombinase in the recruitment of TERRA by strand invasion. Together, our findings reveal a RAD51-dependent pathway that governs TERRA-mediated R-loop formation after transcription, providing a mechanism for the recruitment of lncRNAs to new loci in trans

The makings of TERRA R-loops at chromosome ends

Telomeres protect chromosome ends from nucleolytic degradation, uncontrolled recombination by DNA repair enzymes and checkpoint signaling, and they provide mechanisms for their maintenance by semiconservative DNA replication, telomerase and homologous recombination. The telomeric long noncoding RNA TERRA is transcribed from a large number of chromosome ends. TERRA has been implicated in modulating telomeric chromatin structure and checkpoint signaling, and in telomere maintenance by homology directed repair, and telomerase - when telomeres are damaged or very short. Recent work indicates that TERRA association with telomeres involves the formation of DNA:RNA hybrid structures that can be formed post transcription by the RAD51 DNA recombinase, which in turn may trigger homologous recombination between telomeric repeats and telomere elongation. In this review, we describe the mechanisms of TERRA recruitment to telomeres, R-loop formation and its regulation by shelterin proteins. We discuss the consequences of R-loop formation, with regard to telomere maintenance by DNA recombination and how this may impinge on telomere replication while counteracting telomere shortening in normal cells and in ALT cancer cells, which maintain telomeres in the absence of telomerase