Refined secondary-structure models of the core of yeast and human telomerase RNAs directed by SHAPE

Niederer, Rachel O.; Zappulla, David C.

doi:10.1261/rna.048959.114

Cited by 19 publications

(23 citation statements)

References 59 publications

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“…A similar alternative structure for free human TER was also proposed based on sequence analysis [19], and this structure is consistent with smFRETdata [51]. SHAPE analysis supports pseudoknot folding for S. cerevisiae and human TER t/PK [52]. A recent study using in vivo chemical probing (DMS) also apparently showed that the pseudoknot does form in the protein-free human TER, but the template is sequestered and becomes more exposed on TERT binding [53].…”

Section: Ter Structure and Assembly With Tertsupporting

confidence: 63%

Structural biology of telomerase and its interaction at telomeres

Wang

Feigon

2017

Current Opinion in Structural Biology

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Telomerase is an RNP that synthesizes the 3′ ends of linear chromosomes and is an important regulator of telomere length. It contains a single long non-coding telomerase RNA (TER), telomerase reverse transcriptase (TERT), and other proteins that vary among organisms. Recent progress in structural biology of telomerase includes reports of the first cryo-electron microscopy structure of telomerase, from Tetrahymena, new crystal structures of TERT domains, telomerase RNA structures and models, and identification in Tetrahymena telomerase holoenzyme of human homologues of telomere-associated proteins that have provided a more unified view of telomerase interaction at telomeres as well as insights into the role of telomerase RNA in activity and assembly.

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Section: Ter Structure and Assembly With Tertsupporting

confidence: 63%

Structural biology of telomerase and its interaction at telomeres

Wang

Feigon

2017

Current Opinion in Structural Biology

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“…The pseudoknot of the conserved catalytic center of hTR comprises over half of the core domain, and contains base triples at the junction between the paired stems ( Fig. 2A and B) (14,35,36). In budding yeast, analogous base triples have been shown to contribute directly to catalysis through an unknown mechanism and are located within cross-linking distance from the template (13).…”

Section: Resultsmentioning

confidence: 99%

“…Interestingly, the circular permutation at nucleotide 173 leads to robust activity (Fig. 2C, lane 18), even though it is located within newly proposed base triples (35).…”

Section: Resultsmentioning

confidence: 99%

Physical Connectivity Mapping by Circular Permutation of Human Telomerase RNA Reveals New Regions Critical for Activity and Processivity

Mefford

Zappulla

2016

Molecular and Cellular Biology

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Telomerase is a specialized ribonucleoprotein complex that extends the 3= ends of chromosomes to counteract telomere shortening. However, increased telomerase activity is associated with ϳ90% of human cancers. The telomerase enzyme minimally requires an RNA (hTR) and a specialized reverse transcriptase protein (TERT) for activity in vitro. Understanding the structurefunction relationships within hTR has important implications for human disease. For the first time, we have tested the physicalconnectivity requirements in the 451-nucleotide hTR RNA using circular permutations, which reposition the 5= and 3= ends. Our extensive in vitro analysis identified three classes of hTR circular permutants with altered function. First, circularly permuting 3= of the template causes specific defects in repeat-addition processivity, revealing that the template recognition element found in ciliates is conserved in human telomerase RNA. Second, seven circular permutations residing within the catalytically important core and CR4/5 domains completely abolish telomerase activity, unveiling mechanistically critical portions of these domains. Third, several circular permutations between the core and CR4/5 significantly increase telomerase activity. Our extensive circular permutation results provide insights into the architecture and coordination of human telomerase RNA and highlight where the RNA could be targeted for the development of antiaging and anticancer therapeutics. Linear eukaryotic chromosomes terminate in repeated DNA sequences, called telomeres, which are bound by specific proteins to protect the ends from degradation and detrimental end joining. However, these termini present an end-replication problem that most eukaryotes overcome by utilizing the ribonucleoprotein (RNP) complex telomerase. Telomerase comprises an RNA (hTR in humans) and a reverse transcriptase (TERT), which catalyzes telomere addition. It has been shown that telomeres shorten with aging, and telomerase upregulation occurs in ϳ90% of human cancers (1). Furthermore, mutations in telomerase components have been linked to a variety of short-telomere syndromes, such as dyskeratosis congenita, pulmonary fibrosis, and aplastic anemia (2). Thus, understanding the structure-function relationships of human telomerase RNA is crucial to combat a range of human ailments.During the telomerase catalytic cycle, a short region of the telomerase RNA, known as the template, pairs with the laggingstrand telomeric 3= overhang. The template is then used to direct the iterative addition of the telomeric repeats (TTAGGG in humans), catalyzed by an active site within the TERT protein. Once TERT reaches the end of the RNA template, the DNA substrate is realigned so that additional repeats can be added (3). This ability of the RNA-protein enzyme complex to translocate underlies the enzyme's repeat-addition processivity (RAP). In addition to hTR and TERT, additional accessory proteins bind telomerase in vivo (4). Many studies have been done to identify important regions within hT...

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“…the secondary structure model of the entire 181-nt Est1 arm of TLC1 (nucleotides 514-694) in vitro. We used selective 2 ′ -hydroxyl acylation analyzed by primer extension (SHAPE) to chemically interrogate physical flexibility at each position of purified in vitro-transcribed RNA (Wilkinson et al 2006) using methods described for the catalytic core of yeast and human telomerase RNAs (Niederer and Zappulla 2015). Nucleotides predicted to comprise apical loops of the two hairpins and the 5-nt bulge in the secondary structure model were highly reactive, showing that they are flexible and suggesting that they are indeed unpaired ( …”

Section: Shape Analysis Of the Est1 Arm Of Tlc1 Rnamentioning

confidence: 99%

“…2A). SHAPE was performed as recently described (Niederer and Zappulla 2015). Of note, 2 pmol of purified TLC1 Est1-arm RNA suspended in 8 μL 0.5× TE was denatured at 95°C for 2 min, then cooled on ice for 3 min.…”

Section: Cdc13-est2 Fusion-protein Experimentsmentioning

confidence: 99%

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

Lebo

Niederer

Zappulla

2015

RNA

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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.

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Refined secondary-structure models of the core of yeast and human telomerase RNAs directed by SHAPE

Cited by 19 publications

References 59 publications

Structural biology of telomerase and its interaction at telomeres

Structural biology of telomerase and its interaction at telomeres

Physical Connectivity Mapping by Circular Permutation of Human Telomerase RNA Reveals New Regions Critical for Activity and Processivity

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

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