Ku can contribute to telomere lengthening in yeast at multiple positions in the telomerase RNP

Unlike ribonucleoprotein complexes that have a highly ordered overall architecture, such as the ribosome, yeast telomerase appears to be much more loosely constrained. Here, we investigate the importance of positioning of the Ku subunit within the 1157-nt yeast telomerase RNA (TLC1). Deletion of the 48-nt Ku-binding hairpin in TLC1 RNA (tlc1Δ48) reduces telomere length, survival of cells with gross chromosomal rearrangements, and de novo telomere addition at a broken chromosome end. To test the function of Ku at novel positions in the telomerase RNP, we reintroduced its binding site into tlc1Δ48 RNA at position 446 or 1029. We found that Ku bound to these repositioned sites in vivo and telomere length increased slightly, but statistically significantly. The ability of telomerase to promote survival of cells with gross chromosomal rearrangements by healing damaged chromosome arms was also partially restored, whereas the kinetics of DNA addition to a specific chromosome break was delayed. Having two Ku sites in TLC1 caused progressive hyperelongation of a variable subset of telomeres, consistent with Ku's role in telomerase recruitment to chromosome ends. The number of Ku-binding sites in TLC1 contributed to telomerase RNA abundance in vivo but was only partially responsible for telomere length phenotypes. Thus, telomerase RNA levels and telomere length regulation can be modulated by the number of Ku sites in telomerase RNA. Furthermore, there is substantial flexibility in the relative positioning of Ku in the telomerase RNP for native telomere length maintenance, although not as much flexibility as for the essential Est1p subunit.     

A second essential function of the Est1-binding arm of yeast telomerase RNA

The enzymatic ribonucleoprotein telomerase maintains telomeres in many eukaryotes, including humans, and plays a central role in aging and cancer. Saccharomyces cerevisiae telomerase RNA, TLC1, is a flexible scaffold that tethers telomerase holoenzyme protein subunits to the complex. Here we test the hypothesis that a lengthy conserved region of the Est1-binding TLC1 arm contributes more than simply Est1-binding function. We separated Est1 binding from potential other functions by tethering TLC1 to Est1 via a heterologous RNA-protein binding module. We find that Est1-tethering rescues in vivo function of telomerase RNA alleles missing nucleotides specifically required for Est1 binding, but not those missing the entire conserved region. Notably, however, telomerase function is restored for this condition by expressing the arm of TLC1 in trans. Mutational analysis shows that the Second Essential Est1-arm Domain (SEED) maps to an internal loop of the arm, which SHAPE chemical mapping and 3D modeling suggest could be regulated by conformational change. Finally, we find that the SEED has an essential, Est1-independent role in telomerase function after telomerase recruitment to the telomere. The SEED may be required for establishing telomere extendibility or promoting telomerase RNP holoenzyme activity.     

A miniature yeast telomerase RNA functions in vivo and reconstitutes activity in vitro

The ribonucleoprotein enzyme telomerase synthesizes DNA at the ends of chromosomes. Although the telomerase catalytic protein subunit (TERT) is well conserved, the RNA component is rapidly evolving in both size and sequence. Here, we reduce the 1,157-nucleotide (nt) Saccharomyces cerevisiae TLC1 RNA to a size smaller than the 451-nt human RNA while retaining function in vivo. We conclude that long protein-binding arms are not essential for the RNA to serve its scaffolding function. Although viable, cells expressing Mini-T have shortened telomeres and reduced fitness as compared to wild-type cells, suggesting why the larger RNA has evolved. Previous attempts to reconstitute telomerase activity in vitro using TLC1 and yeast TERT (Est2p) have been unsuccessful. We find that substitution of Mini-T for wild-type TLC1 in a reconstituted system yields robust activity, allowing the contributions of individual yeast telomerase components to be directly assessed.     

Cell cycle-dependent regulation of yeast telomerase by Ku

The heterodimeric Ku complex affects telomere structure in diverse organisms. We report here that in the absence of Ku, the catalytic subunit of telomerase, Est2p, was not telomere-associated in G1 phase, and its association in late S phase was decreased. The telomere association of Est1p, a telomerase component that binds telomeres only in late S phase, was also reduced in the absence of Ku. The effects of Ku on telomerase binding require a 48-nucleotide (nt) stem-loop region of TLC1 telomerase RNA. Ku interacts with TLC1 RNA via this 48-nt region throughout the cell cycle, but this interaction was reduced after telomere replication. These data support a model in which Ku recruits telomerase to telomeres in G1 phase when telomerase is inactive and promotes telomerase-mediated telomere lengthening in late S phase.     

The interaction between the yeast telomerase RNA and the Est1 protein requires three structural elements

In the budding yeast Saccharomyces cerevisiae, the telomerase enzyme is composed of a 1.3-kb TLC1 RNA that forms a complex with Est2 (the catalytic subunit) and two regulatory proteins, Est1 and Est3. Previous work has identified a conserved 5-nt bulge, present in a long helical arm of TLC1, which mediates binding of Est1 to TLC1. However, increased expression of Est1 can bypass the consequences of removal of this RNA bulge, indicating that there are additional binding site(s) for Est1 on TLC1. We report here that a conserved single-stranded internal loop immediately adjacent to the bulge is also required for the Est1-RNA interaction; furthermore, a TLC1 variant that lacks this internal loop but retains the bulge cannot be suppressed by Est1 overexpression, arguing that the internal loop may be a more critical element for Est1 binding. An additional structural feature consisting of a single-stranded region at the base of the helix containing the bulge and internal loop also contributes to recognition of TLC1 by Est1, potentially by providing flexibility to this helical arm. Association of Est1 with each of these TLC1 motifs was assessed using a highly sensitive biochemical assay that simultaneously monitors the relative levels of the Est1 and Est2 proteins in the telomerase complex. The identification of three elements of TLC1 that are required for Est1 association provides a detailed view of this particular protein-RNA interaction.     

RNase III-dependent regulation of yeast telomerase

In bakers' yeast, in vivo telomerase activity requires a ribonucleoprotein (RNP) complex with at least four associated proteins (Est2p, Est1p, Est3p, and Cdc13p) and one RNA species (Tlc1). The function of telomerase in maintaining chromosome ends, called telomeres, is tightly regulated and linked to the cell cycle. However, the mechanisms that regulate the expression of individual components of telomerase are poorly understood. Here we report that yeast RNase III (Rnt1p), a double-stranded RNA-specific endoribonuclease, regulates the expression of telomerase subunits and is required for maintaining normal telomere length. Deletion or inactivation of RNT1 induced the expression of Est1, Est2, Est3, and Tlc1 RNAs and increased telomerase activity, leading to elongation of telomeric repeat tracts. In silico analysis of the different RNAs coding for the telomerase subunits revealed a canonical Rnt1p cleavage site near the 3' end of Est1 mRNA. This predicted structure was cleaved by Rnt1p and its disruption abolished cleavage in vitro. Mutation of the Rnt1p cleavage signal in vivo impaired the cell cycle-dependent degradation of Est1 mRNA without affecting its steady-state level. These results reveal a new mechanism that influences telomeres length by controlling the expression of the telomerase subunits.     

A phylogenetically based secondary structure for the yeast telomerase RNA

BACKGROUND: Telomerase is a ribonucleoprotein complex whose RNA moiety dictates the addition of specific simple sequences onto chromosomes ends. While relevant for certain human genetic diseases, the contribution of the essential telomerase RNA to RNP assembly still remains unclear. Phylogenetic analyses of vertebrate and ciliate telomerase RNAs revealed conserved elements that potentially organize protein subunits for RNP function. In contrast, the yeast telomerase RNA could not be fitted to any known structural model, and the limited number of known sequences from Saccharomyces species did not permit the prediction of a yeast specific conserved structure. RESULTS: We cloned and analyzed the complete telomerase RNA loci (TLC1) from all known Saccharomyces species belonging to the "sensu stricto" group. Complementation analyses in S. cerevisiae and end mappings of mature RNAs ensured the relevance of the cloned sequences. By using phylogenetic comparative analysis coupled with in vitro enzymatic probing, we derived a secondary structure prediction of the Saccharomyces cerevisiae TLC1 RNA. This conserved secondary structure prediction includes a central domain that is likely to orchestrate DNA synthesis and at least two accessory domains important for RNA stability and telomerase recruitment. The structure also reveals a potential tertiary interaction between two loops in the central core. CONCLUSIONS: The predicted secondary structure of the TLC1 RNA of S. cerevisiae reveals a distinct folding pattern featuring well-separated but conserved functional elements. The predicted structure now allows for a detailed and rationally designed study to the structure-function relationships within the telomerase RNP-complex in a genetically tractable system.     

TLC1 RNA nucleo-cytoplasmic trafficking links telomerase biogenesis to its recruitment to telomeres

The yeast telomerase holoenzyme, which adds telomeric repeats at the chromosome ends, is composed of the TLC1 RNA and the associated proteins Est1, Est2 and Est3. To study the biogenesis of telomerase in endogenous conditions, we performed fluorescent in situ hybridization on the native TLC1 RNA. We found that the telomerase RNA colocalizes with telomeres in G1- to S-phase cells. Strains lacking any one of the Est proteins accumulate TLC1 RNA in their cytoplasm, indicating that a critical stage of telomerase biogenesis could take place outside of the nucleus. We were able to demonstrate that endogenous TLC1 RNA shuttles between the nucleus and the cytoplasm, in association with the Crm1p exportin and the nuclear importins Mtr10p-Kap122p. Furthermore, nuclear retention of the TLC1 RNA is impaired in the absence of yKu70p, Tel1p or the MRX complex, which recruit telomerase to telomeres. Altogether, our results reveal that the nucleo-cytoplasmic trafficking of the TLC1 RNA is an important step in telomere homeostasis, and link telomerase biogenesis to its recruitment to telomeres.     

Mutually exclusive binding of telomerase RNA and DNA by Ku alters telomerase recruitment model

In Saccharomyces cerevisiae, the Ku heterodimer contributes to telomere maintenance as a component of telomeric chromatin and as an accessory subunit of telomerase. How Ku binding to double-stranded DNA (dsDNA) and to telomerase RNA (TLC1) promotes Ku's telomeric functions is incompletely understood. We demonstrate that deletions designed to constrict the DNA-binding ring of Ku80 disrupt nonhomologous end-joining (NHEJ), telomeric gene silencing, and telomere length maintenance, suggesting that these functions require Ku's DNA end-binding activity. Contrary to the current model, a mutant Ku with low affinity for dsDNA also loses affinity for TLC1 both in?vitro and in?vivo. Competition experiments reveal that wild-type Ku binds dsDNA and TLC1 mutually exclusively. Cells expressing the mutant Ku are deficient in nuclear accumulation of TLC1, as expected from the RNA-binding defect. These findings force reconsideration of the mechanisms by which Ku assists in recruiting telomerase to natural telomeres and broken chromosome ends. PAPERCLIP:     

Two pathways recruit telomerase to Saccharomyces cerevisiae telomeres

The catalytic subunit of yeast telomerase, Est2p, is a telomere associated throughout most of the cell cycle, while the Est1p subunit binds only in late S/G2 phase, the time of telomerase action. Est2p binding in G1/early S phase requires a specific interaction between telomerase RNA (TLC1) and Ku80p. Here, we show that in four telomerase-deficient strains (cdc13-2, est1Ä, tlc1-SD, and tlc1-BD), Est2p telomere binding was normal in G1/early S phase but reduced to about 40–50% of wild type levels in late S/G2 phase. Est1p telomere association was low in all four strains. Wild type levels of Est2p telomere binding in late S/G2 phase was Est1p-dependent and required that Est1p be both telomere-bound and associated with a stem-bulge region in TLC1 RNA. In three telomerase-deficient strains in which Est1p is not Est2p-associated (tlc1-SD, tlc1-BD, and est2Ä), Est1p was present at normal levels but its telomere binding was very low. When the G1/early S phase and the late S/G2 phase telomerase recruitment pathways were both disrupted, neither Est2p nor Est1p was telomere-associated. We conclude that reduced levels of Est2p and low Est1p telomere binding in late S/G2 phase correlated with an est phenotype, while a WT level of Est2p binding in G1 was not sufficient to maintain telomeres. In addition, even though Cdc13p and Est1p interact by two hybrid, biochemical and genetic criteria, this interaction did not occur unless Est1p was Est2p-associated, suggesting that Est1p comes to the telomere only as part of the holoenzyme. Finally, the G1 and late S/G2 phase pathways for telomerase recruitment are distinct and are likely the only ones that bring telomerase to telomeres in wild-type cells.