POT1 recruits and regulates CST-Polα/primase at human telomeres.

Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At telomeres, Polα/primase is bound to Ctc1/Stn1/Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Our findings suggest that POT1 hinge phosphorylation is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/primase in an inactive, autoinhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/primase into an active state that completes telomere replication through fill-in synthesis.

Telomeres: history, health, and hallmarks of aging.

The escalating social and economic burden of an aging world population has placed aging research at center stage. The hallmarks of aging comprise diverse molecular mechanisms and cellular systems that are interrelated and act in concert to drive the aging process. Here, through the lens of telomere biology, we examine how telomere dysfunction may amplify or drive molecular biological processes underlying each hallmark of aging and contribute to development of age-related diseases such as neurodegeneration and cancer. The intimate link of telomeres to aging hallmarks informs preventive and therapeutic interventions designed to attenuate aging itself and reduce the incidence of age-associated diseases.

Recent Advances in Ciliate Biology.

Ciliates are a diverse group of unicellular eukaryotes that vary widely in size, shape, body plan, and ecological niche. Here, we review recent research advances achieved with ciliate models. Studies on patterning and regeneration have been revived in the giant ciliate Stentor, facilitated by modern omics methods. Cryo-electron microscopy and tomography have revolutionized the structural study of complex macromolecules such as telomerase, ribozymes, and axonemes. DNA elimination, gene scrambling, and mating type determination have been deciphered, revealing interesting adaptations of processes that have parallels in other kingdoms of life. Studies of common eukaryotic processes, such as intracellular trafficking, meiosis, and histone modification, reveal conservation as well as unique adaptations in these organisms that are evolutionarily distant from other models. Continual improvement of genetic and molecular tools makes ciliates accessible models for all levels of education and research. Such advances open new avenues of research and highlight the importance of ciliate research.

Long Noncoding RNAs: Molecular Modalities to Organismal Functions.

We have known for decades that long noncoding RNAs (lncRNAs) can play essential functions across most forms of life. The maintenance of chromosome length requires an lncRNA (e.g., hTERC) and two lncRNAs in the ribosome that are required for protein synthesis. Thus, lncRNAs can represent powerful RNA machines. More recently, it has become clear that mammalian genomes encode thousands more lncRNAs. Thus, we raise the question: Which, if any, of these lncRNAs could also represent RNA-based machines? Here we synthesize studies that are beginning to address this question by investigating fundamental properties of lncRNA genes, revealing new insights into the RNA structure-function relationship, determining cis- and trans-acting lncRNAs in vivo, and generating new developments in high-throughput screening used to identify functional lncRNAs. Overall, these findings provide a context toward understanding the molecular grammar underlying lncRNA biology.

Shaping human telomeres: from shelterin and CST complexes to telomeric chromatin organization.

The regulation of telomere length in mammals is crucial for chromosome end-capping and thus for maintaining genome stability and cellular lifespan. This process requires coordination between telomeric protein complexes and the ribonucleoprotein telomerase, which extends the telomeric DNA. Telomeric proteins modulate telomere architecture, recruit telomerase to accessible telomeres and orchestrate the conversion of the newly synthesized telomeric single-stranded DNA tail into double-stranded DNA. Dysfunctional telomere maintenance leads to telomere shortening, which causes human diseases including bone marrow failure, premature ageing and cancer. Recent studies provide new insights into telomerase-related interactions (the 'telomere replisome') and reveal new challenges for future telomere structural biology endeavours owing to the dynamic nature of telomere architecture and the great number of structures that telomeres form. In this Review, we discuss recently determined structures of the shelterin and CTC1-STN1-TEN1 (CST) complexes, how they may participate in the regulation of telomere replication and chromosome end-capping, and how disease-causing mutations in their encoding genes may affect specific functions. Major outstanding questions in the field include how all of the telomere components assemble relative to each other and how the switching between different telomere structures is achieved.

An Activity Switch in Human Telomerase Based on RNA Conformation and Shaped by TCAB1.

Ribonucleoprotein enzymes require dynamic conformations of their RNA constituents for regulated catalysis. Human telomerase employs a non-coding RNA (hTR) with a bipartite arrangement of domains-a template-containing core and a distal three-way junction (CR4/5) that stimulates catalysis through unknown means. Here, we show that telomerase activity unexpectedly depends upon the holoenzyme protein TCAB1, which in turn controls conformation of CR4/5. Cells lacking TCAB1 exhibit a marked reduction in telomerase catalysis without affecting enzyme assembly. Instead, TCAB1 inactivation causes unfolding of CR4/5 helices that are required for catalysis and for association with the telomerase reverse-transcriptase (TERT). CR4/5 mutations derived from patients with telomere biology disorders provoke defects in catalysis and TERT binding similar to TCAB1 inactivation. These findings reveal a conformational "activity switch" in human telomerase RNA controlling catalysis and TERT engagement. The identification of two discrete catalytic states for telomerase suggests an intramolecular means for controlling telomerase in cancers and progenitor cells.

Structural Insights into Yeast Telomerase Recruitment to Telomeres.

Telomerase maintains chromosome ends from humans to yeasts. Recruitment of yeast telomerase to telomeres occurs through its Ku and Est1 subunits via independent interactions with telomerase RNA (TLC1) and telomeric proteins Sir4 and Cdc13, respectively. However, the structures of the molecules comprising these telomerase-recruiting pathways remain unknown. Here, we report crystal structures of the Ku heterodimer and Est1 complexed with their key binding partners. Two major findings are as follows: (1) Ku specifically binds to telomerase RNA in a distinct, yet related, manner to how it binds DNA; and (2) Est1 employs two separate pockets to bind distinct motifs of Cdc13. The N-terminal Cdc13-binding site of Est1 cooperates with the TLC1-Ku-Sir4 pathway for telomerase recruitment, whereas the C-terminal interface is dispensable for binding Est1 in vitro yet is nevertheless essential for telomere maintenance in vivo. Overall, our results integrate previous models and provide fundamentally valuable structural information regarding telomere biology.

Structure of Telomerase with Telomeric DNA.

Telomerase is an RNA-protein complex (RNP) that extends telomeric DNA at the 3' ends of chromosomes using its telomerase reverse transcriptase (TERT) and integral template-containing telomerase RNA (TER). Its activity is a critical determinant of human health, affecting aging, cancer, and stem cell renewal. Lack of atomic models of telomerase, particularly one with DNA bound, has limited our mechanistic understanding of telomeric DNA repeat synthesis. We report the 4.8 Å resolution cryoelectron microscopy structure of active Tetrahymena telomerase bound to telomeric DNA. The catalytic core is an intricately interlocked structure of TERT and TER, including a previously structurally uncharacterized TERT domain that interacts with the TEN domain to physically enclose TER and regulate activity. This complete structure of a telomerase catalytic core and its interactions with telomeric DNA from the template to telomere-interacting p50-TEB complex provides unanticipated insights into telomerase assembly and catalytic cycle and a new paradigm for a reverse transcriptase RNP.

Spliceosome Profiling Visualizes Operations of a Dynamic RNP at Nucleotide Resolution.

Tools to understand how the spliceosome functions in vivo have lagged behind advances in the structural biology of the spliceosome. Here, methods are described to globally profile spliceosome-bound pre-mRNA, intermediates, and spliced mRNA at nucleotide resolution. These tools are applied to three yeast species that span 600 million years of evolution. The sensitivity of the approach enables the detection of canonical and non-canonical events, including interrupted, recursive, and nested splicing. This application of statistical modeling uncovers independent roles for the size and position of the intron and the number of introns per transcript in substrate progression through the two catalytic stages. These include species-specific inputs suggestive of spliceosome-transcriptome coevolution. Further investigations reveal the ATP-dependent discard of numerous endogenous substrates after spliceosome assembly in vivo and connect this discard to intron retention, a form of splicing regulation. Spliceosome profiling is a quantitative, generalizable global technology used to investigate an RNP central to eukaryotic gene expression.

Timing the Landmark Events in the Evolution of Clear Cell Renal Cell Cancer: TRACERx Renal.

Clear cell renal cell carcinoma (ccRCC) is characterized by near-universal loss of the short arm of chromosome 3, deleting several tumor suppressor genes. We analyzed whole genomes from 95 biopsies across 33 patients with clear cell renal cell carcinoma. We find hotspots of point mutations in the 5' UTR of TERT, targeting a MYC-MAX-MAD1 repressor associated with telomere lengthening. The most common structural abnormality generates simultaneous 3p loss and 5q gain (36% patients), typically through chromothripsis. This event occurs in childhood or adolescence, generally as the initiating event that precedes emergence of the tumor's most recent common ancestor by years to decades. Similar genomic changes drive inherited ccRCC. Modeling differences in age incidence between inherited and sporadic cancers suggests that the number of cells with 3p loss capable of initiating sporadic tumors is no more than a few hundred. Early development of ccRCC follows well-defined evolutionary trajectories, offering opportunity for early intervention.

Telomerase Mechanism of Telomere Synthesis.

Telomerase is the essential reverse transcriptase required for linear chromosome maintenance in most eukaryotes. Telomerase supplements the tandem array of simple-sequence repeats at chromosome ends to compensate for the DNA erosion inherent in genome replication. The template for telomerase reverse transcriptase is within the RNA subunit of the ribonucleoprotein complex, which in cells contains additional telomerase holoenzyme proteins that assemble the active ribonucleoprotein and promote its function at telomeres. Telomerase is distinct among polymerases in its reiterative reuse of an internal template. The template is precisely defined, processively copied, and regenerated by release of single-stranded product DNA. New specificities of nucleic acid handling that underlie the catalytic cycle of repeat synthesis derive from both active site specialization and new motif elaborations in protein and RNA subunits. Studies of telomerase provide unique insights into cellular requirements for genome stability, tissue renewal, and tumorigenesis as well as new perspectives on dynamic ribonucleoprotein machines.

Regulation of human telomerase in homeostasis and disease.

Telomerase is a ribonucleoprotein complex, the catalytic core of which includes the telomerase reverse transcriptase (TERT) and the non-coding human telomerase RNA (hTR), which serves as a template for the addition of telomeric repeats to chromosome ends. Telomerase expression is restricted in humans to certain cell types, and telomerase levels are tightly controlled in normal conditions. Increased levels of telomerase are found in the vast majority of human cancers, and we have recently begun to understand the mechanisms by which cancer cells increase telomerase activity. Conversely, germline mutations in telomerase-relevant genes that decrease telomerase function cause a range of genetic disorders, including dyskeratosis congenita, idiopathic pulmonary fibrosis and bone marrow failure. In this Review, we discuss the transcriptional regulation of human TERT, hTR processing, assembly of the telomerase complex, the cellular localization of telomerase and its recruitment to telomeres, and the regulation of telomerase activity. We also discuss the disease relevance of each of these steps of telomerase biogenesis.

TERRA RNA Antagonizes ATRX and Protects Telomeres.

Through an integration of genomic and proteomic approaches to advance understanding of long noncoding RNAs, we investigate the function of the telomeric transcript, TERRA. By identifying thousands of TERRA target sites in the mouse genome, we demonstrate that TERRA can bind both in cis to telomeres and in trans to genic targets. We then define a large network of interacting proteins, including epigenetic factors, telomeric proteins, and the RNA helicase, ATRX. TERRA and ATRX share hundreds of target genes and are functionally antagonistic at these loci: whereas TERRA activates, ATRX represses gene expression. At telomeres, TERRA competes with telomeric DNA for ATRX binding, suppresses ATRX localization, and ensures telomeric stability. Depleting TERRA increases telomerase activity and induces telomeric pathologies, including formation of telomere-induced DNA damage foci and loss or duplication of telomeric sequences. We conclude that TERRA functions as an epigenomic modulator in trans and as an essential regulator of telomeres in cis.

Active Yeast Telomerase Shares Subunits with Ribonucleoproteins RNase P and RNase MRP.

Telomerase is the ribonucleoprotein enzyme that replenishes telomeric DNA and maintains genome integrity. Minimally, telomerase activity requires a templating RNA and a catalytic protein. Additional proteins are required for activity on telomeres in vivo. Here, we report that the Pop1, Pop6, and Pop7 proteins, known components of RNase P and RNase MRP, bind to yeast telomerase RNA and are essential constituents of the telomerase holoenzyme. Pop1/Pop6/Pop7 binding is specific and involves an RNA domain highly similar to a protein-binding domain in the RNAs of RNase P/MRP. The results also show that Pop1/Pop6/Pop7 function to maintain the essential components Est1 and Est2 on the RNA in vivo. Consistently, addition of Pop1 allows for telomerase activity reconstitution with wild-type telomerase RNA in vitro. Thus, the same chaperoning module has allowed the evolution of functionally and, remarkably, structurally distinct RNPs, telomerase, and RNases P/MRP from unrelated progenitor RNAs.

Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres.

Telomerase maintains genome integrity by adding repetitive DNA sequences to the chromosome ends in actively dividing cells, including 90% of all cancer cells. Recruitment of human telomerase to telomeres occurs during S-phase of the cell cycle, but the molecular mechanism of the process is only partially understood. Here, we use CRISPR genome editing and single-molecule imaging to track telomerase trafficking in nuclei of living human cells. We demonstrate that telomerase uses three-dimensional diffusion to search for telomeres, probing each telomere thousands of times each S-phase but only rarely forming a stable association. Both the transient and stable association events depend on the direct interaction of the telomerase protein TERT with the telomeric protein TPP1. Our results reveal that telomerase recruitment to telomeres is driven by dynamic interactions between the rapidly diffusing telomerase and the chromosome end.

Molecular Profiling Reveals Biologically Discrete Subsets and Pathways of Progression in Diffuse Glioma.

Therapy development for adult diffuse glioma is hindered by incomplete knowledge of somatic glioma driving alterations and suboptimal disease classification. We defined the complete set of genes associated with 1,122 diffuse grade II-III-IV gliomas from The Cancer Genome Atlas and used molecular profiles to improve disease classification, identify molecular correlations, and provide insights into the progression from low- to high-grade disease. Whole-genome sequencing data analysis determined that ATRX but not TERT promoter mutations are associated with increased telomere length. Recent advances in glioma classification based on IDH mutation and 1p/19q co-deletion status were recapitulated through analysis of DNA methylation profiles, which identified clinically relevant molecular subsets. A subtype of IDH mutant glioma was associated with DNA demethylation and poor outcome; a group of IDH-wild-type diffuse glioma showed molecular similarity to pilocytic astrocytoma and relatively favorable survival. Understanding of cohesive disease groups may aid improved clinical outcomes.

CST-Polα/Primase: the second telomere maintenance machine.

It has been known for decades that telomerase extends the 3' end of linear eukaryotic chromosomes and dictates the telomeric repeat sequence based on the template in its RNA. However, telomerase does not mitigate sequence loss at the 5' ends of chromosomes, which results from lagging strand DNA synthesis and nucleolytic processing. Therefore, a second enzyme is needed to keep telomeres intact: DNA polymerase α/Primase bound to Ctc1-Stn1-Ten1 (CST). CST-Polα/Primase maintains telomeres through a fill-in reaction that replenishes the lost sequences at the 5' ends. CST not only serves to maintain telomeres but also determines their length by keeping telomerase from overelongating telomeres. Here we discuss recent data on the evolution, structure, function, and recruitment of mammalian CST-Polα/Primase, highlighting the role of this complex and telomere length control in human disease.

A platform for rapid exploration of aging and diseases in a naturally short-lived vertebrate.

VIDEO ABSTRACT: Aging is a complex process that affects multiple organs. Modeling aging and age-related diseases in the lab is challenging because classical vertebrate models have relatively long lifespans. Here, we develop the first platform for rapid exploration of age-dependent traits and diseases in vertebrates, using the naturally short-lived African turquoise killifish. We provide an integrative genomic and genome-editing toolkit in this organism using our de-novo-assembled genome and the CRISPR/Cas9 technology. We mutate many genes encompassing the hallmarks of aging, and for a subset, we produce stable lines within 2-3 months. As a proof of principle, we show that fish deficient for the protein subunit of telomerase exhibit the fastest onset of telomere-related pathologies among vertebrates. We further demonstrate the feasibility of creating specific genetic variants. This genome-to-phenotype platform represents a unique resource for studying vertebrate aging and disease in a high-throughput manner and for investigating candidates arising from human genome-wide studies.

Early telomerase inactivation accelerates aging independently of telomere length.

Telomerase is required for long-term telomere maintenance and protection. Using single budding yeast mother cell analyses we found that, even early after telomerase inactivation (ETI), yeast mother cells show transient DNA damage response (DDR) episodes, stochastically altered cell-cycle dynamics, and accelerated mother cell aging. The acceleration of ETI mother cell aging was not explained by increased reactive oxygen species (ROS), Sir protein perturbation, or deprotected telomeres. ETI phenotypes occurred well before the population senescence caused late after telomerase inactivation (LTI). They were morphologically distinct from LTI senescence, were genetically uncoupled from telomere length, and were rescued by elevating dNTP pools. Our combined genetic and single-cell analyses show that, well before critical telomere shortening, telomerase is continuously required to respond to transient DNA replication stress in mother cells and that a lack of telomerase accelerates otherwise normal aging.