Single-molecule imaging of telomerase RNA reveals a Recruitment – Retention model for telomere elongation

Laprade, Hadrien; Querido, Emmanuelle; Smith, Michael J.; Guérit, David; Crimmins, Hannah; Conomos, Dimitri; Pourret, E.; Chartrand, Pascal; Sfeir, Agnel

doi:10.1101/2020.01.31.929026

Cited by 9 publications

(21 citation statements)

References 63 publications

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“…Recently, a study showed that the binding of telomeraseassociated protein TCAB1 to hTR positively influences the folding of the P6.1 and P6b stems in an hTR construct lacking the template/pseudoknot domain (Chen et al 2018). Thus, TCAB1 may enforce proper CR4/5 folding either through direct protein-RNA interactions or potentially mediating access to other binding partners via trafficking hTR to Cajal Bodies (Laprade et al 2020). Future studies using methods such as DMS-MapSeq will permit investigation of how hTR folds in vivo during various stages of telomerase biogenesis, as well as how RNA modifications affect hTR folding (Zubradt et al 2017).…”

Section: Discussionmentioning

confidence: 99%

Folding heterogeneity in the essential human telomerase RNA three-way junction

Palka

Forino

Hentschel

et al. 2020

RNA

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Telomeres safeguard the genome by suppressing illicit DNA damage responses at chromosome termini. In order to compensate for incomplete DNA replication at telomeres, most continually dividing cells, including many cancers, express the telomerase ribonucleoprotein (RNP) complex. Telomerase maintains telomere length by catalyzing de novo synthesis of short DNA repeats using an internal telomerase RNA (TR) template. TRs from diverse species harbor structurally conserved domains that contribute to RNP biogenesis and function. In vertebrate TRs, the conserved regions 4 and 5 (CR4/5) fold into a three-way junction (TWJ) that binds directly to the telomerase catalytic protein subunit and is required for telomerase function. We have analyzed the structural properties of the human TR (hTR) CR4/5 domain using a combination of in vitro chemical mapping, secondary structural modeling, and single-molecule structural analysis. Our data suggest the essential P6.1 stem loop within CR4/5 is not stably folded in the absence of the telomerase reverse transcriptase in vitro. Rather, the hTR CR4/5 domain adopts a heterogeneous ensemble of conformations. Finally, single-molecule FRET measurements of CR4/5 and a mutant designed to stabilize the P6.1 stem demonstrate that TERT-binding selects for a structural conformation of CR4/5 that is not the dominant state of the TERT-free in vitro RNA ensemble.

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Section: Discussionmentioning

confidence: 99%

Folding heterogeneity in the essential human telomerase RNA three-way junction

Palka

Forino

Hentschel

et al. 2020

RNA

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“…Moreover, UVcrosslinking induces RNA modifications [17] that can alter the binding affinity of RNA to certain RBPs [18] and impair downstream protein analysis [19]. An alternative approach, tagging of endogenous RNA, requires genetic manipulation and may interfere with endogenous RNA function [20]. Therefore, methods to discover endogenous RNA interacting proteins without genetic manipulation are needed.…”

Section: Introductionmentioning

confidence: 99%

In vivo discovery of RNA proximal proteins via proximity-dependent biotinylation

et al. 2021

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RNA molecules function as messenger RNAs (mRNAs) that encode proteins and noncoding transcripts that serve as adaptor molecules, structural components, and regulators of genome organization and gene expression. Their function and regulation are largely mediated by RNA binding proteins (RBPs). Here we present RNA proximity labelling (RPL), an RNA-centric method comprising the endonuclease-deficient Type VI CRISPR-Cas protein dCas13b fused to engineered ascorbate peroxidase APEX2. RPL discovers target RNA proximal proteins in vivo via proximity-based biotinylation. RPL applied to U1 identified proteins involved in both U1 canonical and noncanonical functions. Profiling of poly(A) tail proximal proteins uncovered expected categories of RBPs and provided additional evidence for 5ʹ-3ʹ proximity and unexplored subcellular localizations of poly(A) + RNA. Our results suggest that RPL allows rapid identification of target RNA binding proteins in native cellular contexts, and is expected to pave the way for discovery of novel RNA-protein interactions important for health and disease.

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“…Among these is the TPP1-POT1 ('protection of telomeres 1') sub-complex, which physically interacts with telomerase at the 3' end, opening and closing the telomere to telomerase activity (Hwang et al, 2012;Wang et al, 2007). While TPP1 recruits telomerase to the telomere (de Lange, 2018), POT1 sterically blocks telomerase from access (Gu et al, 2017;Laprade et al, 2020). Dysregulation of POT1 is therefore common among cancers (reviewed in Wu et al, 2020), contributing to inappropriate cellular immortality.…”

Section: Introductionmentioning

confidence: 99%

The telomere regulatory gene POT1 responds to stress and predicts performance in nature: implications for telomeres and life history evolution

Wolf

Sanders

Beltran

et al. 2021

Preprint

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Long telomeres have become nearly synonymous with a variety of fitness-related traits and may be mediators of ecologically relevant variation in life history strategies. Growing evidence suggests that telomere dynamics are more predictive of performance than length itself, but very little work considers how telomere regulatory mechanisms respond to environmental challenges or influence performance in nature. Here, we combine observational and experimental datasets from free-living tree swallows (Tachycineta bicolor) to assess how performance is predicted by the telomere regulatory gene POT1, which encodes a shelterin protein that sterically blocks telomerase from repairing the telomere. First, we show that lower POT1 gene expression was associated with higher female quality, i.e. earlier breeding, and heavier body mass. We next challenged mothers with an immune stressor (lipopolysaccharide injection) that led to ‘sickness’ in mothers and 24h of food restriction in their offspring. While POT1 did not respond to maternal injection, females with lower constitutive gene expression were better able to maintain feeding rates following treatment. Maternal injection also generated a one-day stressor for chicks, who responded with decreased POT1 gene expression and elongated telomeres. Other putatively stress-responsive mechanisms (i.e. glucocorticoids, antioxidants) were not significantly different between control and stress-exposed chicks. Model comparisons indicated that POT1 mRNA abundance was a largely better predictor of performance than telomere dynamics, indicating that telomere regulators may be powerful modulators of variation in life history strategies.

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Single-molecule imaging of telomerase RNA reveals a Recruitment – Retention model for telomere elongation

Cited by 9 publications

References 63 publications

Folding heterogeneity in the essential human telomerase RNA three-way junction

Folding heterogeneity in the essential human telomerase RNA three-way junction

In vivo discovery of RNA proximal proteins via proximity-dependent biotinylation

The telomere regulatory gene POT1 responds to stress and predicts performance in nature: implications for telomeres and life history evolution

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