Telomerase RNA stem terminus element affects template boundary element function, telomere sequence, and shelterin binding.
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ABSTRACT:
SUBMITTER: Webb CJ
PROVIDER: S-EPMC4568702 | biostudies-other | 2015 Sep
REPOSITORIES: biostudies-other
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Project description:Telomere addition by telomerase requires an internal templating sequence located in the RNA subunit of telomerase. The correct boundary definition of this template sequence is essential for the proper addition of the nucleotide repeats. Incorporation of incorrect telomeric repeats onto the ends of chromosomes has been shown to induce chromosomal instability in ciliate, yeast and human cells. A 5' template boundary defining element (TBE) has been identified in human, yeast and ciliate telomerase RNAs. Here, we report the solution structure of the TBE element (helix II) from Tetrahymena thermophila telomerase RNA. Our results indicate that helix II and its capping pentaloop form a well-defined structure including unpaired, stacked adenine nucleotides in the stem and an unusual syn adenine nucleotide in the loop. A comparison of the T.thermophila helix II pentaloop with a pentaloop of the same sequence found in the 23S rRNA of the Haloarcula marismortui ribosome suggests possible RNA and/or protein interactions for the helix II loop within the Tetrahymena telomerase holoenzyme.
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Project description:Telomerase adds telomeric repeat sequences to chromosome ends using a short region of its RNA subunit as a template. Telomerase RNA subunits are phylogenetically highly divergent, and different strategies have evolved to demarcate the boundary of the template region. The recent identification of the gene encoding telomerase RNA in the fission yeast Schizosaccharomyces pombe (ter1+) has opened the door for structure-function analyses in a model that shares many features with the telomere maintenance machinery of higher eukaryotes. Here we describe a structural element in TER1 that defines the 5' boundary of the template. Disruption of a predicted long range base pairing interaction between template-adjacent nucleotides and a sequence further upstream resulted in reverse transcription beyond the template region and caused telomere shortening. Normal telomere length was restored by combining complementary nucleotide substitutions in both elements, showing that base pairing, not a specific sequence, limits reverse transcription beyond the template. The template boundary described here resembles that of budding yeasts and some mammalian telomerases. However, unlike any previously characterized boundary element, part of the paired region overlaps with the template itself, thus necessitating disruption of these interactions during most reverse transcription cycles. We show that changes in the paired region directly affect the length of individual telomeric repeat units. Our data further illustrate that marginal alignment of the telomeric 3' end with RNA sequences downstream of the template is responsible for primer slippage, causing incorporation of strings of guanosines at the start of a subset of repeats.
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Project description:Human telomerase is a RNA-protein complex that extends the 3' end of linear chromosomes by synthesizing multiple copies of the telomeric repeat TTAGGG1. Its activity is a determinant of cancer progression, stem cell renewal and cellular aging2-5. Telomerase is recruited to telomeres and activated for telomere repeat synthesis by the telomere shelterin protein TPP16,7. Human telomerase has a bilobal structure with a catalytic core ribonuclear protein and a H and ACA box ribonuclear protein8,9. Here we report cryo-electron microscopy structures of human telomerase catalytic core of telomerase reverse transcriptase (TERT) and telomerase RNA (TER (also known as hTR)), and of telomerase with the shelterin protein TPP1. TPP1 forms a structured interface with the TERT-unique telomerase essential N-terminal domain (TEN) and the telomerase RAP motif (TRAP) that are unique to TERT, and conformational dynamics of TEN-TRAP are damped upon TPP1 binding, defining the requirements for recruitment and activation. The structures further reveal that the elements of TERT and TER that are involved in template and telomeric DNA handling-including the TEN domain and the TRAP-thumb helix channel-are largely structurally homologous to those in Tetrahymena telomerase10, and provide unique insights into the mechanism of telomerase activity. The binding site of the telomerase inhibitor BIBR153211,12 overlaps a critical interaction between the TER pseudoknot and the TERT thumb domain. Numerous mutations leading to telomeropathies13,14 are located at the TERT-TER and TEN-TRAP-TPP1 interfaces, highlighting the importance of TER-TERT and TPP1 interactions for telomerase activity, recruitment and as drug targets.
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Project description:The stability of chromosome ends, the telomeres, is dependent on the ribonucleoprotein telomerase. In vitro, telomerase requires at least one RNA molecule and a reverse transcriptase-like protein. However, for telomere homeostasis in vivo, additional proteins are required. Telomerase RNAs of different species vary in size and sequence and only few features common to all telomerases are known. Here we show that stem-loop IVc of the Saccharomyces cerevisiae telomerase RNA contains a structural element that is required for telomerase function in vivo. Indeed, the distal portion of stem-loop IVc stimulates telomerase activity in vitro in a way that is independent of Est1 binding on more proximal portions of this stem-loop. Functional analyses of the RNA in vivo reveal that this distal element we call telomerase-stimulating structure (TeSS) must contain a bulged area in single stranded form and also show that Est1-dependent functions such as telomerase import or recruitment are not affected by TeSS. This study thus uncovers a new structural telomerase RNA element implicated in catalytic activity. Given previous evidence for TeSS elements in ciliate and mammalian RNAs, we speculate that this substructure is a conserved feature that is required for optimal telomerase holoenzyme function.
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Project description:Telomerase is required to maintain repetitive G-rich telomeric DNA sequences at chromosome ends. To do so, the telomerase reverse transcriptase (TERT) subunit reiteratively uses a small region of the integral telomerase RNA (TER) as a template. An essential feature of telomerase catalysis is the strict definition of the template boundary to determine the precise TER nucleotides to be reverse transcribed by TERT. We report the 3-Å crystal structure of the Tetrahymena TERT RNA-binding domain (tTRBD) bound to the template boundary element (TBE) of TER. tTRBD is wedged into the base of the TBE RNA stem-loop, and each of the flanking RNA strands wraps around opposite sides of the protein domain. The structure illustrates how the tTRBD establishes the template boundary by positioning the TBE at the correct distance from the TERT active site to prohibit copying of nontemplate nucleotides.
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Project description:The reverse transcriptase telomerase adds telomeric repeats to chromosome ends to counteract telomere shortening and thereby assures genomic stability in dividing human cells. Key parameters in telomere homeostasis are the frequency with which telomerase engages the chromosome end and the number of telomeric repeats it adds during each association event. To study telomere elongation in vivo, we have established a live-cell imaging assay to track individual telomerase ribonucleoproteins in CRISPR-edited HeLa cells. Using this assay and the drug imetelstat, which is a competitive inhibitor of telomeric DNA binding, we demonstrate that stable association of telomerase with the single-stranded overhang of the chromosome end requires telomerase-DNA base pairing. Furthermore, we show that telomerase processivity contributes to telomere elongation in vivo. Together, these findings provide new insight into the dynamics of telomerase recruitment and the importance of processivity in maintaining telomere length in human cancer cells.
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Project description:Human telomerase RNA (hTR) contains several guanine tracts at its 5'-end that can form a G4-quadruplex structure. Previous evidence suggests that a G4-quadruplex within this region disrupts the formation of an important structure within hTR known as the P1 helix, a critical element in defining the template boundary for reverse transcription. RNA associated with AU-rich element (RHAU) is an RNA helicase that has specificity for DNA and RNA G4-quadruplexes. Two recent studies identify a specific interaction between hTR and RHAU. Herein, we confirm this interaction and identify the minimally interacting RNA fragments. We demonstrate the existence of multiple quadruplex structures within the 5' region of hTR and find that these regions parallel the minimal sequences capable of RHAU interaction. We confirm the importance of the RHAU-specific motif in the interaction with hTR and demonstrate that the helicase activity of RHAU is sufficient to unwind the quadruplex and promote an interaction with 25 internal nucleotides to form a stable P1 helix. Furthermore, we have found that a 5'-terminal quadruplex persists following P1 helix formation that retains affinity for RHAU. Finally, we have investigated the functional implications of this interaction and demonstrated a reduction in average telomere length following RHAU knockdown by small interfering RNA (siRNA).
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Project description:De novo addition of telomeric sequences can occur at broken chromosomes and must be well controlled, which is essential during programmed DNA reorganization processes. In ciliated protozoa an extreme form of DNA-reorganization is observed during macronuclear differentiation after sexual reproduction leading to the elimination of specific parts of the germline genome. Regulating these processes involves small noncoding RNAs, but in addition DNA-reordering, excision and amplification require RNA templates deriving from the parental macronucleus. We show that these putative RNA templates can carry telomeric repeats. Microinjection of RNA templates carrying modified telomeres into the developing macronucleus leads to modified telomeres in vegetative cells, providing strong evidence, that de novo addition of telomeres depends on a telomere-containing transcript from the parental macronucleus.
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Project description:The ends of linear chromosomes are extended by telomerase, a ribonucleoprotein complex minimally consisting of a protein subunit called telomerase reverse transcriptase (TERT) and the telomerase RNA (TER). TERT functions by reverse transcribing a short template region of TER into telomeric DNA. Proper assembly of TERT and TER is essential for telomerase activity; however, a detailed understanding of how TERT interacts with TER is lacking. Previous studies have identified an RNA binding domain (RBD) within TERT, which includes three evolutionarily conserved sequence motifs: CP2, CP, and T. Here, we used site-directed hydroxyl radical probing to directly identify sites of interaction between the TERT RBD and TER, revealing that the CP2 motif is in close proximity to a conserved region of TER known as the template boundary element (TBE). Gel shift assays on CP2 mutants confirmed that the CP2 motif is an RNA binding determinant. Our results explain previous work that established that mutations to the CP2 motif of TERT and to the TBE of TER both permit misincorporation of nucleotides into the growing DNA strand beyond the canonical template. Taken together, these results suggest a model in which the CP2 motif binds the TBE to strictly define which TER nucleotides can be reverse transcribed.
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Project description:The bone marrow failure syndrome dyskeratosis congenita (DC) has been considered to be a disorder of telomere maintenance in which disease features arise due to accelerated shortening of telomeres. By screening core components of the telomerase and shelterin complexes in patients with DC and related bone marrow failure syndromes we have identified 24 novel mutations: 11 in the RNA component of telomerase (TERC), 8 in the reverse transcriptase component (TERT), 4 in dyskerin (DKC1) and 1 in TRF1-interacting nuclear factor 2 (TINF2). This has prompted us to review these genetic subtypes in terms of telomere length, telomerase activity and clinical presentation among 194 genetically characterised index cases recruited onto the registry in London. While those with DKC1 and TINF2 mutations present at a younger age and have more disease features than those with TERC or TERT mutations, there is no difference in telomere length between these groups. There is no difference in the age of onset and numbers of disease features seen in those with TERC and TERT mutations despite the fact that the latter show higher levels of telomerase activity in vitro. The incidence of aplastic anaemia is greater in patients with TERC or TINF2 mutations compared to patients with DKC1 mutations, and cancer incidence is highest in patients with TERC mutations. These data are the first to provide robust comparisons between different genetic subtypes of telomerase and shelterin mutations (the "telomereopathies") and clearly demonstrate that disease severity is not explained by telomere length alone.
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