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To achieve replicative immortality, cancer cells must activate telomere maintenance mechanisms. In 10 to 15% of cancers, this is enabled by recombination-based alternative lengthening of telomeres pathways (ALT). ALT cells display several hallmarks including heterogeneous telomere length, extrachromosomal telomeric repeats, and ALT-associated PML bodies. ALT cells also have high telomeric replication stress (RS) enhanced by fork-stalling structures (R-loops and G4s) and altered chromatin states. In ALT cells, telomeric RS promotes telomere elongation but above a certain threshold becomes detrimental to cell survival. Manipulating RS at telomeres has thus been proposed as a therapeutic strategy against ALT cancers. Through analysis of genome-wide CRISPR fitness screens, we identified ALT-specific vulnerabilities and describe here our characterization of the roles of SUB1, a ssDNA-binding protein, in telomere stability. SUB1 depletion increases RS at ALT telomeres, profoundly impairing ALT cell growth without impacting telomerase-positive cells. During RS, SUB1 is recruited to stalled forks and ALT telomeres via its ssDNA-binding domain. This recruitment is potentiated by RPA depletion, suggesting that these factors may compete for ssDNA. The viability of ALT cells and their resilience toward RS also requires ssDNA binding by SUB1. SUB1 depletion accelerates cell death induced by FANCM depletion, triggering unsustainable levels of telomeric damage in ALT cells. Finally, combining SUB1 depletion with RS-inducing drugs rapidly induces replication catastrophe in ALT cells. Altogether, our work identifies SUB1 as an ALT susceptibility with roles in the mitigation of RS at ALT telomeres and suggests advanced therapeutic strategies for a host of still poorly managed cancers., Competing Interests: Competing interests statement:The authors declare no competing interest.

Telomeric DNA, composed of short, direct repeats, is of crucial importance for chromosome stability. Due to intrinsic problems with replicating this DNA, the repeat tracts shorten at each cell division. Once repeat tracts become critically short, a telomeric stress signal induces cellular senescence and division arrest, which eventually may lead to devastating age-related degenerative diseases associated with dysfunctional telomers. Conversely, maintenance of telomere length by telomerase upregulation is a hallmark of cancer. Therefore, telomere length is a critical determinant of telomere function. How telomere length is established and molecular mechanisms for telomere-specific length regulation remained unknown. Here we show that subtelomeric chromatin is a determinant for how telomere equilibrium set-length is established in cis . The results demonstrate that telomerase recruitment mediated by the telomere-associated Sir4 protein is modulated on chromosome 3L in a telomere-specific way. Increased Sir4 abundance on subtelomeric heterochromatin of this specific telomere leads to telomere lengthening of only that telomere in cis , but not at other telomeres. Therefore, this work describes a mechanism for a how telomere-specific repeat tract length can be established. Further, our results will force the evaluation of telomere length away from a generalized view to a more telomere-specific consideration., Competing Interests: CONFLICT OF INTEREST The authors declare no conflict of interest.

Telomerase, the ribonucleoprotein (RNP) responsible for telomere maintenance, has a complex life. Complex in that it is made of multiple proteins and an RNA, and complex because it undergoes many changes, and passes through different cell compartments. As such, many methods have been developed to discover telomerase components, delve deep into understanding its structure and function and to figure out how telomerase biology ultimately relates to human health and disease. While some old gold-standard methods are still key for determining telomere length and measuring telomerase activity, new technologies are providing promising new ways to gain detailed information that we have never had access to before. Therefore, we thought it timely to briefly review the methods that have revealed information about the telomerase RNP and outline some of the remaining questions that could be answered using new methodology., (© 2023. The Author(s).)

The telomerase ribonucleoprotein particle (RNP) replenishes telomeric DNA and minimally requires an RNA component and a catalytic protein subunit. However, telomerase RNP maturation is an intricate process occurring in several subcellular compartments and is incompletely understood. Here, we report how the co-transcriptional association of key telomerase components and nuclear export factors leads to an export-competent, but inactive, RNP. Export is dependent on the 5' cap, the 3' extension of unprocessed telomerase RNA, and protein associations. When the RNP reaches the cytoplasm, an extensive protein swap occurs, the RNA is trimmed to its mature length, and the essential catalytic Est2 protein joins the RNP. This mature and active complex is then reimported into the nucleus as its final destination and last processing steps. The irreversible processing events on the RNA thus support a ratchet-type model of telomerase maturation, with only a single nucleo-cytoplasmic cycle that is essential for the assembly of mature telomerase., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Alternative splicing (AS) of RNA molecules is a key contributor to transcriptome diversity. In humans, 90%-95% of multi-exon genes produce alternatively spliced RNA transcripts. Therefore, every single gene has the opportunity of producing multiple splice variants, including long non-coding RNA (lncRNA) genes that undergo RNA maturation steps such as conventional and alternative splicing. Emerging evidence suggests significant roles for these lncRNA splice variants in many aspects of cell biology. Differential changes in expression of specific lncRNA splice variants have also been associated with many diseases including cancer. This review covers the current knowledge on this emerging topic of investigation. We provide exclusive insights on the AS landscape of lncRNAs and also describe at the molecular level the functional relevance of lncRNA splice variants, i.e., RNA-based differential functions, production of micropeptides, and generation of circular RNAs. Finally, we discuss exciting perspectives for this emerging field and outline the work required to further develop research endeavors in this field., Competing Interests: The authors have declared no competing interest., (© 2023 The Author(s).)

The incorporation of histone variant H2A.Z into nucleosomes creates specialized chromatin domains that regulate DNA-templated processes, such as gene transcription. In Saccharomyces cerevisiae, the diverging H2A.Z C terminus is thought to provide the H2A.Z exclusive functions. To elucidate the roles of this H2A.Z C terminus genome-wide, we used derivatives in which the C terminus was replaced with the corresponding region of H2A (ZA protein), or the H2A region plus a transcriptional activating peptide (ZA-rII'), with the intent of regenerating the H2A.Z-dependent regulation globally. The distribution of these H2A.Z derivatives indicates that the H2A.Z C-terminal region is crucial for both maintaining the occupation level of H2A.Z and the proper positioning of targeted nucleosomes. Interestingly, the specific contribution on incorporation efficiency versus nucleosome positioning varies enormously depending on the locus analyzed. Specifically, the role of H2A.Z in global transcription regulation relies on its C-terminal region. Remarkably, however, this mostly involves genes without a H2A.Z nucleosome in the promoter. Lastly, we demonstrate that the main chaperone complex which deposits H2A.Z to gene regulatory region (SWR1-C) is necessary to localize all H2A.Z derivatives at their specific loci, indicating that the differential association of these derivatives is not due to impaired interaction with SWR1-C. IMPORTANCE We provide evidence that the Saccharomyces cerevisiae C-terminal region of histone variant H2A.Z can mediate its special function in performing gene regulation by interacting with effector proteins and chaperones. These functional interactions allow H2A.Z not only to incorporate to very specific gene regulatory regions, but also to facilitate the gene expression process. To achieve this, we used a chimeric protein which lacks the native H2A.Z C-terminal region but contains an acidic activating region, a module that is known to interact with components of chromatin-remodeling entities and/or transcription modulators. We reasoned that because this activating region can fulfill the role of the H2A.Z C-terminal region, at least in part, the role of the latter would be to interact with these activating region targets.

Ultraviolet light causes DNA lesions that are removed by nucleotide excision repair (NER). The efficiency of NER is conditional to transcription and chromatin structure. UV induced photoproducts are repaired faster in the gene transcribed strands than in the non-transcribed strands or in transcriptionally inactive regions of the genome. This specificity of NER is known as transcription-coupled repair (TCR). The discovery of pervasive non-coding RNA transcription (ncRNA) advocates for ubiquitous contribution of TCR to the repair of UV photoproducts, beyond the repair of active gene-transcribed strands. Chromatin rules transcription, and telomeres form a complex structure of proteins that silences nearby engineered ectopic genes. The essential protective function of telomeres also includes preventing unwanted repair of double-strand breaks. Thus, telomeres were thought to be transcriptionally inert, but more recently, ncRNA transcription was found to initiate in subtelomeric regions. On the other hand, induced DNA lesions like the UV photoproducts must be recognized and repaired also at the ends of chromosomes. In this study, repair of UV induced DNA lesions was analyzed in the subtelomeric regions of budding yeast. The T4-endonuclease V nicking-activity at cyclobutene pyrimidine dimer (CPD) sites was exploited to monitor CPD formation and repair. The presence of two photoproducts, CPDs and pyrimidine (6,4)-pyrimidones (6-4PPs), was verified by the effective and precise blockage of Taq DNA polymerase at these sites. The results indicate that UV photoproducts in silenced heterochromatin are slowly repaired, but that ncRNA transcription enhances NER throughout one subtelomeric element, called Y', and in distinct short segments of the second, more conserved element, called X. Therefore, ncRNA-transcription dependent TCR assists global genome repair to remove CPDs and 6-4PPs from subtelomeric DNA., Competing Interests: The authors have declared that no competing interests exist.