Rnr1's role in telomere elongation cannot be replaced by Rnr3: a role beyond dNTPs?

Telomeres, the nucleoprotein complexes at the end of eukaryotic chromosomes, protect them from degradation and ensure the replicative capacity of cells. In most human tumors and in budding yeast, telomere length is maintained by the activity of telomerase, an enzyme that adds dNTPs according to an internal RNA template. The dNTPs are generated with the help of the ribonucleotide reductase (RNR) complex. We have recently generated strains lacking the large subunit of RNR, Rnr1, which were kept viable by the expression of RNR complexes containing the Rnr1 homolog, Rnr3. Interestingly, we found that these Rnr1-deficient strains have short telomeres that are stably maintained, but cannot become efficiently elongated by telomerase. Thus, a basic maintenance of short telomeres is possible under conditions, where Rnr1 activity is absent, but a sustained elongation of short telomeres fully depends on Rnr1 activity. We show that Rnr3 cannot compensate for this telomeric function of Rnr1 even when overall cellular dNTP values are restored. This suggests that Rnr1 plays a role in telomere elongation beyond increasing cellular dNTP levels. Furthermore, our data indicate that telomerase may act in two different modes, one that is capable of coping with the "end-replication problem" and is functional even in the absence of Rnr1 and another required for the sustained elongation of short telomeres, which fully depends on the presence of Rnr1. Supply of dNTPs for telomere elongation is provided by the Mec1ATR checkpoint, both during regular DNA replication and upon replication fork stalling. We discuss the implications of these results on telomere maintenance in yeast and cancer cells.

Rnr1, but not Rnr3, facilitates the sustained telomerase-dependent elongation of telomeres.

Ribonucleotide reductase (RNR) provides the precursors for the generation of dNTPs, which are required for DNA synthesis and repair. Here, we investigated the function of the major RNR subunits Rnr1 and Rnr3 in telomere elongation in budding yeast. We show that Rnr1 is essential for the sustained elongation of short telomeres by telomerase. In the absence of Rnr1, cells harbor very short, but functional, telomeres, which cannot become elongated by increased telomerase activity or by tethering of telomerase to telomeres. Furthermore, we demonstrate that Rnr1 function is critical to prevent an early onset of replicative senescence and premature survivor formation in telomerase-negative cells but dispensable for telomere elongation by Homology-Directed-Repair. Our results suggest that telomerase has a "basal activity" mode that is sufficient to compensate for the "end-replication-problem" and does not require the presence of Rnr1 and a different "sustained activity" mode necessary for the elongation of short telomeres, which requires an upregulation of dNTP levels and dGTP ratios specifically through Rnr1 function. By analyzing telomere length and dNTP levels in different mutants showing changes in RNR complex composition and activity we provide evidence that the Mec1ATR checkpoint protein promotes telomere elongation by increasing both dNTP levels and dGTP ratios through Rnr1 upregulation in a mechanism that cannot be replaced by its homolog Rnr3.

Telomere Length Determines TERRA and R-Loop Regulation through the Cell Cycle.

Maintenance of a minimal telomere length is essential to prevent cellular senescence. When critically short telomeres arise in the absence of telomerase, they can be repaired by homology-directed repair (HDR) to prevent premature senescence onset. It is unclear why specifically the shortest telomeres are targeted for HDR. We demonstrate that the non-coding RNA TERRA accumulates as HDR-promoting RNA-DNA hybrids (R-loops) preferentially at very short telomeres. The increased level of TERRA and R-loops, exclusively at short telomeres, is due to a local defect in RNA degradation by the Rat1 and RNase H2 nucleases, respectively. Consequently, the coordination of TERRA degradation with telomere replication is altered at shortened telomeres. R-loop persistence at short telomeres contributes to activation of the DNA damage response (DDR) and promotes recruitment of the Rad51 recombinase. Thus, the telomere length-dependent regulation of TERRA and TERRA R-loops is a critical determinant of the rate of replicative senescence.

Mixed Integer Linear Programming based machine learning approach identifies regulators of telomerase in yeast.

Understanding telomere length maintenance mechanisms is central in cancer biology as their dysregulation is one of the hallmarks for immortalization of cancer cells. Important for this well-balanced control is the transcriptional regulation of the telomerase genes. We integrated Mixed Integer Linear Programming models into a comparative machine learning based approach to identify regulatory interactions that best explain the discrepancy of telomerase transcript levels in yeast mutants with deleted regulators showing aberrant telomere length, when compared to mutants with normal telomere length. We uncover novel regulators of telomerase expression, several of which affect histone levels or modifications. In particular, our results point to the transcription factors Sum1, Hst1 and Srb2 as being important for the regulation of EST1 transcription, and we validated the effect of Sum1 experimentally. We compiled our machine learning method leading to a user friendly package for R which can straightforwardly be applied to similar problems integrating gene regulator binding information and expression profiles of samples of e.g. different phenotypes, diseases or treatments.

Breaking new ground: digging into TERRA function.

Despite the fact that telomeres carry chromatin marks typically associated with silent heterochromatin, they are actively transcribed into TElomeric Repeat containing RNA (TERRA). TERRA transcription is conserved from yeast to man, initiates in the subtelomeric region and proceeds through the telomeric tract of presumably each individual telomere. TERRA levels are increased in yeast survivors and in cancer cells employing ALT as a telomere maintenance mechanism (TMM). Thus, TERRA may be a promising biomarker and potential target in anti-cancer therapy. Interestingly, several recent publications implicate TERRA in regulatory processes including telomere end protection and the establishment of the heterochromatic state at telomeres. A picture is emerging whereby TERRA acts as a regulator of telomere length and hence the associated onset of replicative senescence in a cell. In this review we will summarize the latest results regarding TERRA transcription, localization and related function. A special focus will be set on the potential role of TERRA in the regulation of telomere length and replicative senescence. Possible implications of increased TERRA levels in yeast survivors and in ALT cancer cells will be discussed.

Telomeric RNA-DNA hybrids affect telomere-length dynamics and senescence.

Although telomeres are heterochromatic, they are transcribed into noncoding telomeric repeat-containing RNA (TERRA). Here we show that RNA-DNA hybrids form at telomeres and are removed by RNase H enzymes in the budding yeast, Saccharomyces cerevisiae. In recombination-competent telomerase mutants, telomeric RNA-DNA hybrids promote recombination-mediated elongation events that delay the onset of cellular senescence. Reduction of TERRA and telomeric RNA-DNA-hybrid levels diminishes rates of recombination-mediated telomere elongation in cis. Overexpression of RNase H decreases telomere recombination rates and accelerates senescence in recombination-competent but not recombination-deficient cells. In contrast, in the absence of both telomerase and homologous recombination, accumulation of telomeric RNA-DNA hybrids leads to telomere loss and accelerated rates of cellular senescence. Therefore, the regulation of TERRA transcription and telomeric RNA-DNA-hybrid formation are important determinants of both telomere-length dynamics and proliferative potential after the inactivation of telomerase.

Telomeres and disease: enter TERRA.

Telomere function is tightly regulated in order to maintain chromosomal stability. When telomeres become dysfunctional, the replicative capacity of cells diminishes and cellular senescence ensues. This can lead to impaired tissue replenishment and eventually degenerative disorders, referred to as telomere syndromes. Cancer can also develop as a result of the genomic instability associated with telomere dysfunction. TERRA (TElomeric Repeat containing RNA) is a long non-coding transcript that stems from sub-telomeric regions and continues into the telomeric tract and is therefore a hybrid of both sub-telomeric and telomeric sequence. In general, increased TERRA transcription is associated with telomere shortening and compromised telomere function. Here we will briefly outline the general principles behind telomere dysfunction-associated diseases. Furthermore, we will discuss the few known links that exist between telomere transcription (TERRA) and disease. Finally, we will speculate on how the understanding, and eventual manipulation, of TERRA transcription could potentially be used in terms of therapeutic strategies.

Deregulated telomere transcription causes replication-dependent telomere shortening and promotes cellular senescence.

Telomeres are transcribed into non-coding TElomeric Repeat containing RNAs (TERRA). We have employed a transcriptionally inducible telomere to investigate how telomere transcription affects telomere function in Saccharomyces cerevisiae. We report that telomere shortening resulting from high levels of telomere transcription stems from a DNA replication-dependent loss of telomere tracts, which can occur independent of both telomerase inhibition and homologous recombination. We show that in order for telomere loss to occur, transcription must pass through the telomere tract itself producing a TERRA molecule. We demonstrate that increased telomere transcription of a single telomere leads to a premature cellular senescence in the absence of a telomere maintenance mechanism (telomerase and homology directed repair). Similar rapid senescence and telomere shortening are also seen in sir2Δ cells with compromised telomere maintenance, where TERRA levels are increased at natural telomeres. These data suggest that telomere transcription must be tightly controlled to prevent telomere loss and early onset senescence.