A human-Tetrahymena pseudoknot chimeric telomerase RNA reconstitutes a nonprocessive enzyme in vitro that is defective in telomere elongation

The phylogenetically-derived secondary structures of telomerase RNAs (TR) from ciliates, yeasts and vertebrates are surprisingly conserved and contain a pseudoknot domain at a similar location downstream of the template. As the pseudoknot domains of Tetrahymena TR (tTR) and human TR (hTR) mediate certain similar functions, we hypothesized that they might be functionally interchangeable. We constructed a chimeric TR (htTR) by exchanging the hTR pseudoknot sequences for the tTR pseudoknot region. The chimeric RNA reconstituted human telomerase activity when coexpressed with hTERT in vitro, but exhibited defects in repeat addition processivity and levels of DNA synthesis compared to hTR. Activity was dependent on tTR sequences within the chimeric RNA. htTR interacted with hTERT in vitro and dimerized predominantly via a region of its hTR backbone, the J7b/8a loop. Introduction of htTR in telomerase-negative cells stably expressing hTERT did not reconstitute an active enzyme able to elongate telomeres. Thus, our results indicate that the chimeric RNA reconstituted a weakly active nonprocessive human telomerase enzyme in vitro that was defective in telomere elongation in vivo. This suggests that there may be species-specific requirements for pseudoknot functions.     

Exploring metazoan evolution through dynamic and holistic changes in protein families and domains

ABSTRACT: BACKGROUND: Proteins convey the majority of biochemical and cellular activities in organisms. Over the course of evolution, proteins undergo normal sequence mutations as well as large scale mutations involving domain duplication and/or domain shuffling. These events result in the generation of new proteins and protein families. Processes that affect proteome evolution drive species diversity and adaptation. Herein, change over the course of metazoan evolution, as defined by birth/death and duplication/deletion events within protein families and domains, was examined using the proteomes of 9 metazoan and two outgroup species. RESULTS: In studying members of the three major metazoan groups, the vertebrates, arthropods, and nematodes, we found that the number of protein families increased at the majority of lineages over the course of metazoan evolution where the magnitude of these increases was greatest at the lineages leading to mammals. In contrast, the number of protein domains decreased at most lineages and at all terminal lineages. This resulted in a weak correlation between protein family birth and domain birth; however, the correlation between domain birth and domain member duplication was quite strong. These data suggest that domain birth and protein family birth occur via different mechanisms, and that domain shuffling plays a role in the formation of protein families. The ratio of protein family birth to protein domain birth (domain shuffling index) suggests that shuffling had a more demonstrable effect on protein families in nematodes and arthropods than in vertebrates. Through the contrast of high and low domain shuffling indices at the lineages of Trichinella spiralis and Gallus gallus, we propose a link between protein redundancy and evolutionary changes controlled by domain shuffling; however, the speed of adaptation among the different lineages was relatively invariant. Evaluating the functions of protein families that appeared or disappeared at the last common ancestors (LCAs) of the three metazoan clades supports a correlation with organism adaptation. Furthermore, bursts of new protein families and domains in the LCAs of metazoans and vertebrates are consistent with whole genome duplications. CONCLUSION: Metazoan speciation and adaptation were explored by birth/death and duplication/deletion events among protein families and domains. Our results provide insights into protein evolution and its bearing on metazoan evolution.     

Non-animal origin of animal thioredoxin reductases: implications for selenocysteine evolution and evolution of protein function through carboxy-terminal extensions

Thioredoxin reductase (TR) and thioredoxin constitute a major cellular redox system present in all organisms. In contrast to a single form of thioredoxin, there are two TR types: One (bacterial type or small TR) is present in bacteria, archaea, plants, and most unicellular eukaryotes, whereas the second (animal or large TR) is only found in animals and typically contains a carboxy-terminal penultimate selenocysteine encoded by TGA. Surprisingly, we detected sequences of large TRs in various unicellular eukaryotes. Moreover, green algae Chlamydomonas reinhardtii had both small and large TRs, with the latter being a selenoprotein, but no examples of horizontal gene transfer from animals to the green algae could be detected. In addition, phylogenetic analyses revealed that large TRs formed a subgroup of lower eukaryotic glutathione reductases (GRs). The data suggest that the large TR evolved in a lower eukaryote capable of selenocysteine insertion rather than in an animal. The enzyme appeared to evolve by a carboxy-terminal extension of GR such that the resulting carboxy-terminal glutathionelike peptide became an intramolecular substrate for GR and a reductant for thioredoxin. Subsequently, small TRs were lost in an organism that gave rise to animals, large TRs were lost in plants and fungi, and selenocysteine/cysteine replacements took place in some large TRs. Our data implicate carboxy-terminal extension of proteins as a general mechanism of evolution of new protein function.     

Secondary structure of vertebrate telomerase RNA

Telomerase is a ribonucleoprotein enzyme that maintains telomere length by adding telomeric sequence repeats onto chromosome ends. The essential RNA component of telomerase provides the template for telomeric repeat synthesis. To determine the secondary structure of vertebrate telomerase RNA, 32 new telomerase RNA genes were cloned and sequenced from a variety of vertebrate species including 18 mammals, 2 birds, 1 reptile, 7 amphibians, and 4 fishes. Using phylogenetic comparative analysis, we propose a secondary structure that contains four structural domains conserved in all vertebrates. Ten helical regions of the RNA are universally conserved while other regions vary significantly in length and sequence between different classes of vertebrates. The proposed vertebrate telomerase RNA structure displays a strikingly similar topology to the previously determined ciliate telomerase RNA structure, implying an evolutionary conservation of the global architecture of telomerase RNA.     

A Cajal body-independent pathway for telomerase trafficking in mice

The intranuclear trafficking of human telomerase involves a dynamic interplay between multiple nuclear sites, most notably Cajal bodies and telomeres. Cajal bodies are proposed to serve as sites of telomerase maturation, storage, and assembly, as well as to function in the cell cycle-regulated delivery of telomerase to telomeres in human cells. Here, we find that telomerase RNA does not localize to Cajal bodies in mouse cells, and instead resides in separate nuclear foci throughout much of the cell cycle. However, as in humans, mouse telomerase RNA (mTR) localizes to subsets of telomeres specifically during S phase. The localization of mTR to telomeres in mouse cells does not require coilin-containing Cajal bodies, as mTR is found at telomeres at similar frequencies in cells from wild-type and coilin knockout mice. At the same time, we find that human TR localizes to Cajal bodies (as well as telomeres) in mouse cells, indicating that the distinct trafficking of mTR is attributable to an intrinsic property of the RNA (rather than a difference in the mouse cell environment such as the properties of mouse Cajal bodies). We also find that during S phase, mTR foci coalesce into short chains, with at least one of the conjoined mTR foci co-localizing with a telomere. These findings point to a novel, Cajal body-independent pathway for telomerase biogenesis and trafficking in mice.     

Molecular Evolution of TEPP Protein Genes in Metazoans

TEPP is a gene expressed in human reproductive organs such as testis, prostate, and placenta. Here, identification and molecular evolutionary analysis of TEPP proteins in various metazoan animals including deuterostomes (chordates, hemichordates, and echinoderms), lophotrochozoans (mollusks and annelids), and cnidarians (sea anemone and coral) are reported. A multiple sequence alignment revealed two highly conserved regions in TEPP proteins that had no similarity to any other known domains or proteins. Genomic sequence analysis showed frequent shifting of the splice sites of intron 1 in mammalian TEPP genes. A comparison of the intron positions in the coding region showed that the exon/intron structure of the TEPP gene was established in an early metazoan ancestor and that independent loss of a single intron occurred in echinoderms and in vertebrates. The urochordate tunicate TEPP genes are intronless, possibly due to replacement of the original gene by a retrogene. No homolog was detected in birds, insects, nematodes, and teleost fishes despite the extensive sequence data of these species, implying that the TEPP gene might be lost in these lineages.     

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.     

Amphioxus postembryonic development reveals the homology of chordate metamorphosis

Most studies in evolution are centered on how homologous genes, structures, and/or processes appeared and diverged. Although historical homology is well defined as a concept, in practice its establishment can be problematic, especially for some morphological traits or developmental processes. Metamorphosis in chordates is such an enigmatic character. Defined as a spectacular postembryonic larva-to-adult transition, it shows a wide morphological diversity between the different chordate lineages, suggesting that it might have appeared several times independently. In vertebrates, metamorphosis is triggered by binding of the thyroid hormones (THs) T(4) and T(3) to thyroid-hormone receptors (TRs). Here we show that a TH derivative, triiodothyroacetic acid (TRIAC), induces metamorphosis in the cephalochordate amphioxus. The amphioxus TR (amphiTR) mediates spontaneous and TRIAC-induced metamorphosis because it strongly binds to TRIAC, and a specific TR antagonist, NH3, inhibits both spontaneous and TRIAC-induced metamorphosis. Moreover, as in amphibians, amphiTR expression levels increase around metamorphosis and are enhanced by THs. Therefore, TH-regulated metamorphosis, mediated by TR, is an ancestral feature of all chordates. This conservation of a regulatory network supports the homology of metamorphosis in the chordate lineage.