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 the correct distance from the TERT active site to prohibit copying of non-template nucleotides.

Telomerase reverse transcribes short guanine (G)-rich DNA repeat sequences from its internal RNA template to maintain telomere length. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays, we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using GQ-stabilizing K+ versus GQ-destabilizing Li+ salts yielded changes in DNA product profiles consistent with formation of GQ structures within the telomerase–DNA complex. Addition of the telomerase processivity factor POT1–TPP1 altered the DNA product profile, but was not sufficient to recover full activity in the presence of Li+ cations. This result suggests GQ folding synergizes with POT1–TPP1 to support telomerase function. Single-molecule Förster resonance energy transfer experiments reveal complex DNA structural dynamics during real-time catalysis in the presence of K+ but not Li+, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we globally fit telomerase time-series data to a kinetic model that converges to a set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis, and provide a detailed characterization of GQ modulation of polymerase function.

Rapamycin is a naturally occurring macrolide whose target is at the core of nutrient and stress regulation in a wide range of species. Despite well-established roles as an inhibitor of cap-dependent mRNA translation, relatively little is known about its effects on other modes of RNA processing. Here, we characterize the landscape of rapamycin-induced post-transcriptional gene regulation. Transcriptome analysis of rapamycin-treated cells reveals genome-wide changes in alternative mRNA splicing and pronounced changes in NMD-sensitive isoforms. We demonstrate that despite well-documented attenuation of cap-dependent mRNA translation, rapamycin can augment NMD of certain transcripts. Rapamycin-treatment significantly reduces the levels of both endogenous and exogenous Premature Termination Codon (PTC)-containing mRNA isoforms and its effects are dose-, UPF1- and 4EBP-dependent. The PTC-containing SRSF6 transcript exhibits a shorter half-life upon rapamycin-treatment as compared to the non-PTC isoform. Rapamycin-treatment also causes depletion of PTC-containing mRNA isoforms from polyribosomes, underscoring the functional relationship between translation and NMD. Enhanced NMD activity also correlates with an enrichment of the nuclear Cap Binding Complex (CBC) in rapamycin-treated cells. Our data demonstrate that rapamycin modulates global RNA homeostasis by NMD.

Telomerase maintains telomere length by reverse transcribing short G-rich DNA repeat sequences from its internal RNA template. G-rich telomere DNA repeats readily fold into Gquadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using K + versus Li + salts yield changes in DNA product profiles consistent with formation of GQ structure within the telomerase-DNA complex. Single-molecule FRET experiments reveal complex DNA structural dynamics during real-time catalysis, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we fit telomerase time series data to a global kinetic model that converges to a unique set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis and provide a detailed characterization of GQ modulation of polymerase function. SIGNIFICANCETelomeres protect the ends of linear chromosomes from illicit DNA processing events that can threaten genome stability. Telomere structure is built upon repetitive G-rich DNA repeat sequences that have the ability to fold into stable secondary structures called G-quadruplexes (GQs). In rapidly dividing cells, including the majority of human cancers, telomeres are maintained by the specialized telomerase enzyme. Thus, telomerase and its telomere DNA substrates represent important targets for developing novel cancer drugs. In this work, we provide evidence for GQ folding within the newly synthesized DNA product of an actively extending telomerase enzyme. Our results highlight the delicate interplay between the structural properties of telomere DNA and telomerase function.

Telomerase is an ancient ribonucleoprotein (RNP) that protects the ends of linear chromosomes from the loss of critical coding sequences through repetitive addition of short DNA sequences. These repeats comprise the telomere, which together with many accessory proteins, protect chromosomal ends from degradation and unwanted DNA repair. Telomerase is a unique reverse transcriptase (RT) that carries its own RNA to use as a template for repeat addition. Over decades of research, it has become clear that there are many diverse, crucial functions played by telomerase RNA beyond simply acting as a template. In this review, we highlight recent findings in three model systems: ciliates, yeast and vertebrates, that have shifted the way the field views the structural and mechanistic role(s) of RNA within the functional telomerase RNP complex. Viewed in this light, we hope to demonstrate that while telomerase RNA is just one example of the myriad functional RNA in the cell, insights into its structure and mechanism have wide-ranging impacts. WIREs RNA 2018, 9:e1456. doi: 10.1002/wrna.1456 This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution.

The original discovery of enzymes that synthesize DNA using an RNA template appeared to contradict the central dogma of biology, in which information is transferred, in a unidirectional way, from DNA genes into RNA molecules. The paradigm-shifting discovery of RNA-dependent DNA polymerases, also called reverse transcriptases (RTs), reshaped existing views for how cells function; however, the scope of the impact RTs impose on biology had yet to be realized. In the decades of research since the early 1970s, the biomedical and biotechnological significance of retroviral RTs, as well as the evolutionarily related telomerase enzyme, has become exceedingly clear. One common theme that has emerged in the course of RT-related research is the central role of nucleic acid binding and dynamics during enzyme function. However, directly interrogating these dynamic properties is challenging because of the stochastic properties of biological macromolecules. In this review, we describe how the development of single-molecule biophysical techniques has opened new windows through which to observe the dynamic behavior of this remarkable class of enzymes. Specifically, we focus on how the powerful single-molecule Förster resonance energy transfer (FRET) method has been exploited to study the structure and function of the human immunodeficiency virus (HIV) RT and telomerase ribonucleoprotein (RNP) enzymes. These exciting studies have refined our understanding of RT catalysis, have revealed unforeseen structural rearrangements between RTs and their nucleic acid substrates, and have helped to characterize the mode of action of RT-inhibiting drugs. We conclude with a discussion of how the ongoing development of single-molecule technologies will continue to empower researchers to probe RT mechanisms in new and exciting ways. Outline 1 Introduction 2 Implications of RT discoveries 3 On the evolution of viral RTs and telomerase4 Single-molecule methods to study RT enzymes 5 Human immunodeficiency virus (HIV) RT 6 Conformational dynamics of HIV RT on nucleic acids 7 RT sliding, substrate recognition, and strand displacement synthesis 8 Structural dynamics of the HIV RT initiation complex 9 Telomerase 10 Structural dynamics of Tetrahymena thermophila telomerase 11 Nucleic acid dynamics during human telomerase catalysis 12 Conclusions and perspectives References

RNA surveillance by the Nonsense Mediated Decay (NMD) pathway eliminates potentially deleterious transcripts containing Premature Termination Codons (PTCs). The transition from a pioneering round of translation to steady state translation is hypothesized to be a major checkpoint in this process. One hallmark of mRNAs licensed for translation is the exchange of 7-methylguanosine cap binding proteins. However, mRNAs undergoing steady state translation are also NMD substrates, raising mechanistic questions about the NMD checkpoint. To test the role of cap binding proteins in NMD, we modulated the protein composition of cytoplasmic messenger ribonucleoprotein particles (mRNPs) with the naturally occurring macrolide rapamycin. We demonstrate that despite well-documented attenuation of cap-dependent mRNA translation, rapamycin can augment NMD. Rapamycin-treatment significantly reduces the levels of endogenous and exogenous PTC-containing mRNA isoforms in a dose-and UPF1-dependent manner. PTC-containing transcripts exhibit a shorter half-life upon rapamacyin-treatment as compared to non-PTC isoforms. Rapamycin also causes depletion of PTC-containing mRNA isoforms from polyribosomes, suggesting that actively translating ribosomes can transition between low and high NMD states. Importantly, mRNPs show depletion of eIF4E and retention of the nuclear Cap BindingComplex (CBC) in rapamycin-treated cells. Our data demonstrate that rapamycin potentiates pioneer-like mRNP context thereby decreasing NMD evasion.

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