Cell cycle-dependent transcription factors control the expression of yeast telomerase RNA.

Telomerase is a specialized ribonucleoprotein that adds repeated DNA sequences to the ends of eukaryotic chromosomes to preserve genome integrity. Some secondary structure features of the telomerase RNA are very well conserved, and it serves as a central scaffold for the binding of associated proteins. The Saccharomyces cerevisiae telomerase RNA, TLC1, is found in very low copy number in the cell and is the limiting component of the known telomerase holoenzyme constituents. The reasons for this low abundance are unclear, but given that the RNA is very stable, transcriptional control mechanisms must be extremely important. Here we define the sequences forming the TLC1 promoter and identify the elements required for its low expression level, including enhancer and repressor elements. Within an enhancer element, we found consensus sites for Mbp1/Swi4 association, and chromatin immunoprecipitation (ChIP) assays confirmed the binding of Mbp1 and Swi4 to these sites of the TLC1 promoter. Furthermore, the enhancer element conferred cell cycle-dependent regulation to a reporter gene, and mutations in the Mbp1/Swi4 binding sites affected the levels of telomerase RNA and telomere length. Finally, ChIP experiments using a TLC1 RNA-binding protein as target showed cell cycle-dependent transcription of the TLC1 gene. These results indicate that the budding yeast TLC1 RNA is transcribed in a cell cycle-dependent fashion late in G1 and may be part of the S phase-regulated group of genes involved in DNA replication.

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.

Regulation of telomere structure and functions by subunits of the INO80 chromatin remodeling complex.

ATP-dependent chromatin remodeling complexes have been implicated in the regulation of transcription, replication, and more recently DNA double-strand break repair. Here we report that the Ies3p subunit of the Saccharomyces cerevisiae INO80 chromatin remodeling complex interacts with a conserved tetratricopeptide repeat domain of the telomerase protein Est1p. Deletion of IES3 and some other subunits of the complex induced telomere elongation and altered telomere position effect. In telomerase-negative mutants, loss of Ies3p delayed the emergence of recombinational survivors and stimulated the formation of extrachromosomal telomeric circles in survivors. Deletion of IES3 also resulted in heightened levels of telomere-telomere fusions in telomerase-deficient strains. In addition, a delay in survivor formation was observed in an Arp8p-deficient mutant. Because Arp8p is required for the chromatin remodeling activity of the INO80 complex, the complex may promote recombinational telomere maintenance by altering chromatin structure. Consistent with this notion, we observed preferential localization of multiple subunits of the INO80 complex to telomeres. Our results reveal novel functions for a subunit of the telomerase complex and the INO80 chromatin remodeling complex.

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.

Two-dimensional agarose gel analysis of DNA replication intermediates.

The neutral/neutral (N/N) two-dimensional (2-D) agarose gel technique is a useful tool for understanding the mechanisms leading to the complete duplication of linear eukaryotic chromosomes. For the yeast Saccharomyces cerevisiae, it has been used to localize and characterize origins of replication as well as fork progression characteristics in a variety of experimental settings. The method involves running a first-dimension gel in order to separate restriction-digested replication intermediates of different mass. A gel slice containing the continuum of replicating DNA is then cut and subjected to a second-dimension gel, such as to resolve replication intermediates of varying topology. The 2-D gel is then blotted and probed to allow an examination of replication intermediates in specific DNA regions.

Telomerase core components protect Candida telomeres from aberrant overhang accumulation.

Telomerase is a cellular reverse transcriptase that extends one strand (the G-strand) of the telomere terminal repeats. Aside from this role in telomere length maintenance, telomerase has been proposed to serve a protective function at chromosome ends, although this is not well understood mechanistically. Earlier analysis suggests that, in the pathogenic yeast Candida albicans, the catalytic reverse transcriptase subunit of telomerase (TERT/EST2) can protect telomeres against nucleolytic degradation. In this report we demonstrate that the RNA component (TER1) has a similar function; in addition to complete loss of telomerase activity and progressive telomere attrition, the ter1-DeltaDelta strains manifested a dramatic increase in the amount of G-strand overhangs, consistent with aberrant degradation of the complementary C-strand. We also demonstrate that a catalytically incompetent EST2 protein can suppress such overhang accumulation in the est2-DeltaDelta mutant to the same extent as the wild-type protein. Altogether, our data support the notion that the Candida telomerase core components mediate a protective function through a mechanism that is independent of its catalytic activity.