Mutational analysis of the Tetrahymena telomerase RNA: identification of residues affecting telomerase activity in vitro.

Telomere-specific repeat sequences are essential for chromosome end stability. Telomerase maintains telomere length by adding sequences de novo onto chromosome ends. The template domain of the telomerase RNA component dictates synthesis of species-specific telomeric repeats and other regions of the RNA have been suggested to be important for enzyme structure and/or catalysis. Using enzyme reconstituted in vitro with RNAs containing deletions or substitutions we identified nucleotides in the RNA component that are important for telomerase activity. Although many changes to conserved features in the RNA secondary structure did not abolish enzyme activity, levels of activity were often greatly reduced, suggesting that regions other than the template play a role in telomerase function. The template boundary was only altered by changes in stem II that affected the conserved region upstream of the template, not by changes in other regions, such as stems I, III and IV, consistent with a role of the conserved region in defining the 5' boundary of the template. Surprisingly, telomerase RNAs with substitutions or deletion of residues potentially abolishing the conserved pseudoknot structure had wild-type levels of telomerase activity. This suggests that this base pairing interaction may not be required for telomerase activity per se but may be conserved as a regulatory site for the enzyme in vivo.     

A critical three-way junction is conserved in budding yeast and vertebrate telomerase RNAs

The telomerase ribonucleoprotein copies a short template within its integral RNA moiety onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Non-template regions of telomerase RNA (TER) are also crucial for telomerase function, yet they are highly divergent in sequence among species and their roles are largely unclear. Using both phylogenetic and mutational analyses, we predicted secondary structures for TERs from Kluyveromyces budding yeast species. A comparison of these secondary structure models with the published model for the Saccharomyces cerevisiae TER reveals a common arrangement into three long arms, a templating domain in the center and several conserved elements in the same positions within the structure. One of them, a three-way junction element, is highly conserved in budding yeast TERs. This element also shows sequence and structure similarity to the critical CR4-CR5 activating domain of vertebrate TERs. Mutational analysis in Kluyveromyces lactis confirmed that this element, and in particular the residues conserved across yeast and vertebrates, is critical for telomerase action both in vivo and in vitro. These findings demonstrate that despite the extreme divergence of TER sequences from different organisms, they do share conserved elements, which presumably carry out common roles in telomerase function.     

A conserved secondary structure for telomerase RNA.

The RNA moiety of the ribonucleoprotein enzyme telomerase contains the template for telomeric DNA synthesis. We present a secondary structure model for telomerase RNA, derived by a phylogenetic comparative analysis of telomerase RNAs from seven tetrahymenine ciliates. The telomerase RNA genes from Tetrahymena malaccensis, T. pyriformis, T. hyperangularis, T. pigmentosa, T. hegewishii, and Glaucoma chattoni were cloned, sequenced, and compared with the previously cloned RNA gene from T. thermophila and with each other. To define secondary structures of these RNAs, homologous complementary sequences were identified by the occurrence of covariation among putative base pairs. Although their primary sequences have diverged rapidly overall, a strikingly conserved secondary structure was identified for all these telomerase RNAs. Short regions of nucleotide conservation include a block of 22 totally conserved nucleotides that contains the telomeric templating region.     

Ciliate telomerase RNA structural features.

Telomerase RNA is an integral part of telomerase, the ribonucleoprotein enzyme that catalyzes the synthesis of telomeric DNA. The RNA moiety contains a templating domain that directs the synthesis of a species-specific telomeric repeat and may also be important for enzyme structure and/or catalysis. Phylogenetic comparisons of telomerase RNA sequences from various Tetrahymena spp. and hypotrich ciliates have revealed two conserved secondary structure models that share many features. We have cloned and sequenced the telomerase RNA genes from an additional six Tetrahymena spp. (T. vorax, T. borealis, T. australis, T. silvana, T. capricornis and T. paravorax). Inclusion of these sequences, most notably that from T. paravorax, in a phylogenetic comparative analysis allowed us to more narrowly define structural elements that may be necessary for a minimal telomerase RNA. A primary sequence element, positioned 5' of the template and conserved between all previously known ciliate telomerase RNAs, has been reduced from 5'-(C)UGUCA-3' to the 4 nt sequence 5'-GUCA-3'. Conserved secondary structural features and the impact they have on the general organization of ciliate telomerase RNAs is discussed.     

Circular rDNA Replicons Persist in Tetrahymena thermophila Transformants Synthesizing GGGGTC Telomeric Repeats

Site-directed mutagenesis of the telomerase RNA from Tetrahymena thermophila was used previously to demonstrate the templating function of a sequence within this RNA; this sequence specifies the sequence of telomeric DNA in vivo. The possible functional importance of a phylogenetically conserved nucleotide outside the telomerase RNA template region was investigated by a similar experimental approach. The telomerase RNA gene was altered by site-directed mutagenesis, cloned in a circular selectable transformation vector consisting of an rRNA gene carrying a selectable drug resistance marker, and introduced into macronuclei of vegetatively dividing Tetrahymena thermophila by microinjection. Changing an invariant A to U at position 16 of the telomerase RNA (A16U) had no effect detectable by phenotype on telomerase function in vivo. However these experiments showed that a telomerase template alteration that dictates the synthesis of the mutant telomeric DNA sequence GGGGTC leads to a profound change in the population of rDNA replicons. The addition of GGGGTC mutant repeats leads to selective pressure for the loss of high copy linear rDNA, and the rRNA genes are maintained in the form of the circular rDNA replicons introduced during transformation.     

一种含有端粒重复序列非编码RNA的发现及最新研究进展

端粒是染色体末端的特化结构,由简单呈串联线性排列的核酸重复序列及相关蛋白质组成。其核酸序列具有高度的保守性,均富含GC。在人类为TTAGGG的高度重复序列具有维持基因组完整性的作用。端粒功能异常会导致染色体失去稳定性,促进肿瘤的发生和发展。以往认为端粒附近区域不具有转录活性,但最近在Science杂志上Azzalin等首次报道了该区域可以转录一种非编码RNA,即端粒RNA(telomeric RNA)。该分子具有特殊的UUAGGG重复序列,在调控端粒长度和端粒酶活性上具有重要作用,在发育、衰老和肿瘤发生发展等研究中已成为热点。该文将对近期有关端粒RNA的研究进展予以综述。     

Dynamics of telomere erosion in transformed and non-transformed avian cells in vitro

Although vertebrate telomeres are highly conserved, telomere dynamics and telomerase profiles vary among species. The objective of the present study was to examine telomerase activity and telomere length profiles of transformed and non-transformed avian cells in vitro. Non-transformed chicken embryo fibroblasts (CEFs) showed little or no telomerase activity from the earliest passages through senescence. Unexpectedly, a single culture of particularly long-lived senescent CEFs showed telomerase activity after over 250 days in culture. Transformed avian lines (six chicken, two quail and one turkey) and tumor samples (two chicken) exhibited telomerase activity. Telomere length profiles of non-transformed CEF cultures derived from individual embryos of an inbred line (UCD 003) exhibited cycles of shortening and lengthening with a substantial net loss of telomeric DNA by senescence. The telomere length profiles of several transformed cell lines resembled telomere length profiles of senescent CEFs in that they exhibited little of the typical smear of terminal restriction fragments (TRFs) suggesting that these transformed cells may possess a reduced amount of telomeric DNA. These results show that avian telomerase activity profiles are consistent with the telomerase activity profiles of human primary and transformed cells. Further, monitoring of telomere lengths of primary cells provides evidence for a dynamic series of changes over the lifespan of any specific cell culture ultimately resulting in net telomeric DNA loss by senescence.     

Structure of telomeric chromatin in <Emphasis Type="Italic">Drosophila</Emphasis>

The telomeric nucleoprotein complex protects linear chromosome ends from degradation. In contrast to most eukaryotes in which telomerase is responsible for telomere elongation by adding short DNA repeats synthesized using an RNA template, the telomere elongation in Drosophila involves transposition of specialized telomeric retroelements onto chromosome ends. Proteins that bind telomeric and subtelomeric sequences form specific telomeric chromatin, and its components are highly conserved among organisms employing different mechanisms of telomere elongation. This review is focused on the analysis of components of the Drosophila telomeric complex and its comparison with telomeric proteins in telomerase-encoded organisms. Structural and functional analysis of Drosophila telomeres suggests that there are three distinct chromatin regions: protective structure at the very end of chromosome (cap), subtelomeric region which is characterized by condensed chromatin structure, and the terminal retrotransposon array whose expression is under the control of an RNAi (RNA interference)-based mechanism. The link between RNAi and telomeric chromatin formation in germinal tissues is discussed.     

Telomere biology of trypanosomatids: beginning to answer some questions

Studies of telomere structure and maintenance in trypanosomatids have provided insights into the evolutionary origin and conservation of some telomeric components shared by trypanosomes and vertebrates. For example, trypanosomatid telomeres are maintained by telomerase and consist of the canonical TTAGGG repeats, which in Trypanosoma brucei can form telomeric loops (t-loops). However, the telomeric chromatin of trypanosomatids is composed of organism-specific proteins and other proteins that share little sequence similarity with their vertebrate counterparts. Because telomere maintenance mechanisms are essential for genome stability, we propose that the particular features shown by the trypanosome telomeric chromatin hold the key for the design of antiparasitic drugs.