ABSTRACT
Given its central role in life, DNA is remarkably easy to damage. Double strand breaks (DSBs) are the most toxic form of DNA damage, and DSBs pose the greatest danger to genomic integrity. In higher vertebrates, the non-homologous end joining pathway (NHEJ) is the predominate pathway that repairs DSBs. NHEJ has three steps: 1) DNA end recognition by the DNA dependent protein kinase [DNA-PK], 2) DNA end-processing by numerous NHEJ accessory factors, and 3) DNA end ligation by the DNA ligase IV complex (LX4). Although this would appear to be a relatively simple mechanism, it has become increasingly apparent that it is not. Recently, much insight has been derived regarding the mechanism of non-homologous end joining through a proliferation of cryo-EM studies, structure-function mutational experiments informed by these new structural data, and novel single-molecule imaging approaches. An emerging consensus in the field is that NHEJ progresses from initial DSB end recognition by DNA-PK to synapsis of the two DNA ends in a long-range synaptic complex where ends are held too far apart (115â¯Å) for ligation, and then progress to a short-range synaptic complex where ends are positioned close enough for ligation. What was surprising from these structural studies was the observation of two distinct types of DNA-PK dimers that represent NHEJ long-range complexes. In this review, we summarize current knowledge about the function of the distinct NHEJ synaptic complexes and align this new information with emerging cellular single-molecule microscopy studies as well as with previous studies of DNA-PK's function in repair.
Subject(s)
DNA Breaks, Double-Stranded , DNA End-Joining Repair , DNA-Activated Protein Kinase , Humans , DNA-Activated Protein Kinase/metabolism , Animals , DNA/metabolism , Chromosome Pairing , DNA Ligase ATP/metabolismABSTRACT
Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-stranded breaks (DSBs) in vertebrates. However, due to challenges in detecting DSBs in living cells, the repair capacity of the NHEJ pathway is unknown. The DNA termini of many DSBs must be processed to allow ligation while minimizing genetic changes that result from break repair. Emerging models propose that DNA termini are first synapsed ~115Å apart in one of several long-range synaptic complexes before transitioning into a short-range synaptic complex that juxtaposes DNA ends to facilitate ligation. The transition from long-range to short-range synaptic complexes involves both conformational and compositional changes of the NHEJ factors bound to the DNA break. Importantly, it is unclear how NHEJ proceeds in vivo because of the challenges involved in analyzing recruitment of NHEJ factors to DSBs over time in living cells. Here, we develop a new approach to study the temporal and compositional dynamics of NHEJ complexes using live cell single-molecule imaging. Our results provide direct evidence for stepwise maturation of the NHEJ complex, pinpoint key regulatory steps in NHEJ progression, and define the overall repair capacity NHEJ in living cells.
ABSTRACT
Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged organelles. Autophagosome formation starts with autophagy factors accumulating on lipid vesicles containing ATG9. These phagophores attach to donor membranes, expand via ATG2-mediated lipid transfer, capture cargo, and mature into autophagosomes, ultimately fusing with lysosomes for their degradation. Autophagy can be activated by nutrient stress, for example, by a reduction in the cellular levels of amino acids. In contrast, how autophagy is regulated by low cellular ATP levels via the AMP-activated protein kinase (AMPK), an important therapeutic target, is less clear. Using live-cell imaging and an automated image analysis pipeline, we systematically dissect how nutrient starvation regulates autophagosome biogenesis. We demonstrate that glucose starvation downregulates autophagosome maturation by AMPK-mediated inhibition of phagophore tethering to donor membrane. Our results clarify AMPKs regulatory role in autophagy and highlight its potential as a therapeutic target to reduce autophagy.
Subject(s)
AMP-Activated Protein Kinases , Autophagosomes , Autophagy , Humans , AMP-Activated Protein Kinases/metabolism , Autophagosomes/metabolism , Glucose/metabolism , Cell LineABSTRACT
BACKGROUND: Magnetic resonance (MRI) imaging of the skeletal muscles (muscle MRI for short) is increasingly being used in clinical routine for diagnosis and longitudinal assessment of muscle disorders. However, cross-centre standards for measurement protocol and radiological assessment are still lacking. OBJECTIVES: The aim of this expert recommendation is to present standards for the application and interpretation of muscle MRI in hereditary and inflammatory muscle disorders. METHODS: This work was developed in collaboration between neurologists, neuroradiologists, radiologists, neuropaediatricians, neuroscientists and MR physicists from different university hospitals in Germany. The recommendations are based on expert knowledge and a focused literature search. RESULTS: The indications for muscle MRI are explained, including the detection and monitoring of structural tissue changes and oedema in the muscle, as well as the identification of a suitable biopsy site. Recommendations for the examination procedure and selection of appropriate MRI sequences are given. Finally, steps for a structured radiological assessment are presented. CONCLUSIONS: The present work provides concrete recommendations for the indication, implementation and interpretation of muscle MRI in muscle disorders. Furthermore, it provides a possible basis for the standardisation of the measurement protocols at all clinical centres in Germany.
Subject(s)
Magnetic Resonance Imaging , Muscle, Skeletal , Magnetic Resonance Imaging/standards , Magnetic Resonance Imaging/methods , Humans , Germany , Muscle, Skeletal/diagnostic imaging , Muscle, Skeletal/pathology , Muscular Diseases/diagnostic imaging , Practice Guidelines as Topic , Radiology/standards , Neurology/standardsABSTRACT
BACKGROUND: Magnetic resonance (MRI) imaging of the skeletal muscles (muscle MRI for short) is increasingly being used in clinical routine for diagnosis and longitudinal assessment of muscle disorders. However, cross-centre standards for measurement protocol and radiological assessment are still lacking. OBJECTIVES: The aim of this expert recommendation is to present standards for the application and interpretation of muscle MRI in hereditary and inflammatory muscle disorders. METHODS: This work was developed in collaboration between neurologists, neuroradiologists, radiologists, neuropaediatricians, neuroscientists and MR physicists from different university hospitals in Germany. The recommendations are based on expert knowledge and a focused literature search. RESULTS: The indications for muscle MRI are explained, including the detection and monitoring of structural tissue changes and oedema in the muscle, as well as the identification of a suitable biopsy site. Recommendations for the examination procedure and selection of appropriate MRI sequences are given. Finally, steps for a structured radiological assessment are presented. CONCLUSIONS: The present work provides concrete recommendations for the indication, implementation and interpretation of muscle MRI in muscle disorders. Furthermore, it provides a possible basis for the standardisation of the measurement protocols at all clinical centres in Germany.
Subject(s)
Magnetic Resonance Imaging , Muscular Diseases , Magnetic Resonance Imaging/methods , Magnetic Resonance Imaging/standards , Humans , Muscular Diseases/diagnostic imaging , Germany , Practice Guidelines as Topic , Muscle, Skeletal/diagnostic imaging , Muscle, Skeletal/pathologyABSTRACT
Since the publication of the 2013 European Neuromuscular Center (ENMC) diagnostic criteria for Inclusion Body Myositis (IBM), several advances have been made regarding IBM epidemiology, pathogenesis, diagnostic tools, and clinical trial readiness. Novel diagnostic tools include muscle imaging techniques such as MRI and ultrasound, and serological testing for cytosolic 5'-nucleotidase-1A antibodies. The 272nd ENMC workshop aimed to develop new diagnostic criteria, discuss clinical outcome measures and clinical trial readiness. The workshop started with patient representatives highlighting several understudied symptoms and the urge for a timely diagnosis. This was followed by presentations from IBM experts highlighting the new developments in the field. This report is composed of two parts, the first part providing new diagnostic criteria on which consensus was achieved. The second part focuses on the use of outcome measures in clinical practice and clinical trials, highlighting current limitations and outlining the goals for future studies.
Subject(s)
Myositis, Inclusion Body , Myositis , Humans , Consensus , Magnetic Resonance Imaging , Myositis/diagnosis , Myositis, Inclusion Body/therapy , Myositis, Inclusion Body/drug therapy , Netherlands , Outcome Assessment, Health CareABSTRACT
Idiopathic inflammatory myopathies are a group of rare, autoimmune, diseases typically involving striate muscle and also variously affecting several other systems or organs, such as joints, skin, lungs, heart and gastrointestinal tract. IIM are mainly characterised by subacute onset and chronic course and are burdened by significant morbidity and mortality. Despite the rarity of these conditions, several efforts have been undertaken in the last years to better understand their pathogenesis, as well as to achieve a more precise classification and to define the optimal therapeutic approach. The aim of this review is to provide an up-to-date digest of the most relevant studies published on this topic over the last year.
Subject(s)
Autoimmune Diseases , Myositis , Humans , Myositis/diagnosis , Myositis/drug therapy , Autoimmune Diseases/diagnosis , Autoimmune Diseases/therapyABSTRACT
Telomerase is a complex ribonucleoprotein scaffolded by the telomerase RNA (TR). Telomere lengthening by telomerase is essential to maintain the proliferative potential of stem cells and germ cells, and telomerase is inappropriately activated in the majority of cancers. Assembly of TR with its 12 protein co-factors and the maturation of the 5'- and 3'-ends of TR have been the focus of intense research efforts over the past two decades. High-resolution Cryo-EM structures of human telomerase, high-throughput sequencing of the 3' end of TR, and live cell imaging of various telomerase components have significantly advanced our understanding of the molecular mechanisms that govern telomerase biogenesis, yet many important questions remain unaddressed. In this review, we will summarize these recent advances and highlight the remaining key questions with the ultimate goal of targeting telomerase assembly to suppress telomere maintenance in cancer cells or to promote telomerase activity in patients affected by telomere shortening disorders.
Subject(s)
Neoplasms , Telomerase , Humans , Telomerase/metabolism , Telomere/metabolism , Ribonucleoproteins/genetics , Telomere HomeostasisABSTRACT
Adult-onset idiopathic inflammatory myopathy (IIM) is associated with an increased cancer risk within the 3 years preceding and following IIM onset. Evidence- and consensus-based recommendations for IIM-associated cancer screening can potentially improve outcomes. This International Guideline for IIM-Associated Cancer Screening provides recommendations addressing IIM-associated cancer risk stratification, cancer screening modalities and screening frequency. The international Expert Group formed a total of 18 recommendations via a modified Delphi approach using a series of online surveys. First, the recommendations enable an individual patient's IIM-associated cancer risk to be stratified into standard, moderate or high risk according to the IIM subtype, autoantibody status and clinical features. Second, the recommendations outline a 'basic' screening panel (including chest radiography and preliminary laboratory tests) and an 'enhanced' screening panel (including CT and tumour markers). Third, the recommendations advise on the timing and frequency of screening via basic and enhanced panels, according to risk status. The recommendations also advise consideration of upper or lower gastrointestinal endoscopy, nasoendoscopy and 18F-FDG PET-CT scanning in specific patient populations. These recommendations are aimed at facilitating earlier IIM-associated cancer detection, especially in those who are at a high risk, thus potentially improving outcomes, including survival.
Subject(s)
Myositis , Neoplasms , Adult , Humans , Early Detection of Cancer , Positron Emission Tomography Computed Tomography , Neoplasms/diagnosis , Myositis/complications , Myositis/diagnosis , AutoantibodiesABSTRACT
Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged organelles. Autophagosome formation starts with autophagy factors accumulating on lipid vesicles containing ATG9. These phagophores attach to donor membranes, expand via ATG2-mediated lipid transfer, capture cargo, and mature into autophagosomes, ultimately fusing with lysosomes for their degradation. Autophagy can be activated by nutrient stress, for example by a reduction in the cellular levels of amino acids. In contrast, how autophagy is regulated by low cellular ATP levels via the AMP-activated protein kinase (AMPK), an important therapeutic target, is less clear. Using live-cell imaging and an automated image analysis pipeline, we systematically dissect how nutrient starvation regulates autophagosome biogenesis. We demonstrate that glucose starvation downregulates autophagosome maturation by AMPK mediated inhibition of phagophores tethering to donor membranes. Our results clarify AMPK's regulatory role in autophagy and highlight its potential as a therapeutic target to reduce autophagy.
ABSTRACT
BACKGROUND: Myasthenia gravis (MG) affects individuals as a chronic autoimmune disease for many years. Commonly, chronic diseases significantly reduce the patients' quality of life. Aiming to improve the future quality of life in MG, this study assessed the factors impacting quality of life. As gender-specific medicine is becoming increasingly important, this study also focused on understanding gender differences in the outcome of MG. METHODS: The study is a combined monocentric, retrospective and prospective database analysis of patient records based on 2,370 presentations of 165 patients with clinically, serologically and/or electrophysiologically confirmed MG over an observation period of up to 47 years. The data collection included the following parameters: antibody status, disease severity, age, medication use, gender, and disease duration. In addition, a prospective survey was conducted on the quality of life using the Myasthenia gravis-specific 15-item Quality of Life scale (MG-QoL15) and on the activities of daily living using the MG-specific Activities of Daily Living scale (MG-ADL). RESULTS: Of the 165 patients, 85 were male (51.5%) and 80 were female (48.5%). The remaining baseline characteristics (e.g. age and antibody status) were consistent with other myasthenia gravis cohorts. A high body mass index (BMI) (p = 0.005) and a high disease severity (p < 0.001) were significantly associated with lower disease-specific quality of life. Additionally, the quality of life in women with MG was significantly reduced compared to male patients (19.7 vs. 13.0 points in the MG-QoL15, p = 0.024). Gender differences were also observable in terms of the period between initial manifestation and initial diagnosis and women were significantly more impaired in their activities of daily living (MG-ADL) than men (4.8 vs. 3.0 points, p = 0.032). CONCLUSION: Women with MG had significantly poorer disease specific quality of life compared to men as well as patients with a higher BMI. In order to improve the quality of life, gender-specific medicine and further investigation regarding a modification of the quality of life by lowering the BMI are essential and necessary. TRIAL REGISTRATION: Study approval by the Ethics Committee of the University Medical Center Göttingen was granted (number 6/5/18).
Subject(s)
Myasthenia Gravis , Quality of Life , Humans , Male , Female , Activities of Daily Living , Cohort Studies , Overweight/complications , Retrospective Studies , Myasthenia Gravis/complications , Surveys and QuestionnairesABSTRACT
Introduction: We aimed to identify B-cell-mediated immunomechanisms in inclusion body myositis (IBM) and polymyositis (PM) as part of the complex pathophysiology. Materials and methods: Human primary myotube cultures were derived from orthopedic surgery. Diagnostic biopsy specimens from patients with IBM (n=9) and PM (n=9) were analyzed for markers of B cell activation (BAFF and APRIL) and for chemokines that control the recruitment of B cells (CXCL-12 and CXCL-13). Results were compared to biopsy specimens without myopathic changes (n=9) and hereditary muscular dystrophy (n=9). Results: The mRNA expression of BAFF, APRIL, and CXCL-13 was significantly higher in IBM and PM compared to controls. Patients with IBM displayed the highest number of double positive muscle fibers for BAFF and CXCL-12 (48%) compared to PM (25%), muscular dystrophy (3%), and non-myopathic controls (0%). In vitro, exposure of human myotubes to pro-inflammatory cytokines led to a significant upregulation of BAFF and CXCL-12, but APRIL and CXCL-13 remained unchanged. Conclusion: The results substantiate the hypothesis of an involvement of B cell-associated mechanisms in the pathophysiology of IBM and PM. Muscle fibers themselves seem to contribute to the recruitment of B cells and sustain inflammation.
Subject(s)
Myositis, Inclusion Body , Myositis , Polymyositis , Humans , Inflammation , Muscle Fibers, SkeletalABSTRACT
OBJECTIVES: To compare clinical characteristics, including the frequency of cutaneous, extramuscular manifestations, and malignancy, between adults with anti-synthetase syndrome (ASyS) and dermatomyositis (DM). METHODS: Using data regarding adults from the MYONET registry, a cohort of DM patients with anti-Mi2/-TIF1É£/-NXP2/-SAE/-MDA5 autoantibodies, and a cohort of ASyS patients with anti-tRNA synthetase autoantibodies (anti-Jo1/-PL7/-PL12/-OJ/-EJ/-Zo/-KS) were identified. Patients with DM sine dermatitis or with discordant dual autoantibody specificities were excluded. Sub-cohorts of patients with ASyS with or without skin involvement were defined based on presence of DM-type rashes (heliotrope rash, Gottron's papules/sign, violaceous rash, shawl sign, V sign, erythroderma, and/or periorbital rash). RESULTS: In total 1,054 patients were included (DM, n = 405; ASyS, n = 649). In ASyS cohort, 31% (n = 203) had DM-type skin involvement (ASyS-DMskin). A higher frequency of extramuscular manifestations, including Mechanic's hands, Raynaud's phenomenon, arthritis, interstitial lung disease, and cardiac involvement differentiated ASyS-DMskin from DM (all p< 0.001), whereas higher frequency of any of four DM-type rashes: heliotrope rash (n = 248, 61% vs n = 90, 44%), violaceous rash (n = 166, 41% vs n = 57, 9%), V sign (n = 124, 31% vs n = 28, 4%), and shawl sign (n = 133, 33% vs n = 18, 3%) differentiated DM from ASyS-DMskin (all p< 0.005). Cancer-associated myositis (CAM) was more frequent in DM (n = 67, 17%) compared with ASyS (n = 21, 3%) and ASyS-DMskin (n = 7, 3%) cohorts (both p< 0.001). CONCLUSION: DM-type rashes are frequent in patients with ASyS; however, distinct clinical manifestations differentiate these patients from classical DM. Skin involvement in ASyS does not necessitate increased malignancy surveillance. These findings will inform future ASyS classification criteria and patient management.
ABSTRACT
The genetic material in human cells is continuously exposed to a wide variety of insults that can induce different DNA lesions. To maintain genomic stability and prevent potentially deleterious genetic changes caused by DNA damage, mammalian cells have evolved a number of pathways that repair specific types of DNA damage. These DNA repair pathways vary in their accuracy, some providing high-fidelity repair while others are error-prone and are only activated as a last resort. Adding additional complexity to cellular mechanisms of DNA repair is the DNA damage response which is a sophisticated a signaling network that coordinates repair outcomes, cell-cycle checkpoint activation, and cell fate decisions. As a result of the sheer complexity of the various DNA repair pathways and the DNA damage response there are large gaps in our understanding of the molecular mechanisms underlying DNA damage repair in human cells. A key unaddressed question is how the dynamic recruitment of DNA repair factors contributes to repair kinetics and repair pathway choice in human cells. Methodological advances in live cell single-molecule imaging over the last decade now allow researchers to directly observe and analyze the dynamics of DNA repair proteins in living cells with high spatiotemporal resolution. Live cell single-molecule imaging combined with single-particle tracking can provide direct insight into the biochemical reactions that control DNA repair and has the power to identify previously unobservable processes in living cells. This review summarizes the main considerations for experimental design and execution for live cell single-molecule imaging experiments and describes how they can be used to define the molecular mechanisms of DNA damage repair in mammalian cells.
Subject(s)
DNA Repair , Single Molecule Imaging , Humans , DNA , DNA Damage , Signal TransductionABSTRACT
Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are the primary pathways for repairing DNA double-strand breaks (DSBs) during interphase, whereas microhomology-mediated end-joining (MMEJ) has been regarded as a backup mechanism. Through CRISPR-Cas9-based synthetic lethal screens in cancer cells, we identified subunits of the 9-1-1 complex (RAD9A-RAD1-HUS1) and its interacting partner, RHINO, as crucial MMEJ factors. We uncovered an unexpected function for RHINO in restricting MMEJ to mitosis. RHINO accumulates in M phase, undergoes Polo-like kinase 1 (PLK1) phosphorylation, and interacts with polymerase θ (Polθ), enabling its recruitment to DSBs for subsequent repair. Additionally, we provide evidence that MMEJ activity in mitosis repairs persistent DSBs that originate in S phase. Our findings offer insights into the synthetic lethal relationship between the genes POLQ and BRCA1 and BRAC2 and the synergistic effect of Polθ and poly(ADP-ribose) polymerase (PARP) inhibitors.
Subject(s)
Cell Cycle Proteins , DNA Breaks, Double-Stranded , DNA End-Joining Repair , Exonucleases , Mitosis , Humans , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Exonucleases/genetics , Exonucleases/metabolism , HEK293 Cells , Mitosis/genetics , Poly(ADP-ribose) Polymerases/metabolismABSTRACT
Inclusion body myositis (IBM) is a chronic, mostly treatment-resistant, inflammatory myopathy with a pathology that centers around specific interactions between inflammation and protein accumulation. The study aimed to identify the inflammasome as a key event in the complex network of pathomechanisms. Regulation of the inflammasome was assessed in a well-established pro-inflammatory cell culture model using human myoblasts and primary human myotubes. By quantitative PCR, western blot and immunocytochemistry, inflammasome markers including NLRP3 were assessed in muscle cells exposed to the cytokines IL-1ß and IFN-γ. The data were corroborated by analysis of muscle biopsies from patients with IBM compared to other myositis subtypes. In the cell culture model of IBM, the NLRP3 inflammasome was significantly overexpressed, as evidenced by western blot (p = 0.03) and quantitative PCR (p < 0.01). Target genes that play a role in inflammasome assembly, T-cell migration, and MHC-I expression (p = 0.009) were highly co-upregulated. NLRP3 was significantly overexpressed in muscle biopsies from IBM samples compared to disease controls (p = 0.049), including other inflammatory myopathies. Due to the extraordinary features of the pathogenesis and the pronounced upregulation of NLRP3 in IBM, the inflammasome could serve as a key molecule that drives the inflammatory cascade as well as protein accumulation in the muscle. These data can be useful for future therapeutic developments.
Subject(s)
Myositis, Inclusion Body , Myositis , Humans , Myositis, Inclusion Body/metabolism , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Cells, Cultured , Muscle, Skeletal/metabolism , Myositis/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolismABSTRACT
ABBREVIATIONS: ATG: autophagy-related proteins; ULK1/2: Unc-51-Like activating Kinases; PI3Ks: Phosphoinositide 3-Kinases; ATG2A: autophagy-related protein 2A; ATG5: autophagy-related protein 5; ATG16: autophagy-related protein 16; ATG8: autophagy-related protein 8; U2OS: human bone osteosarcoma epithelial cell; LC3B: microtubule-associated protein 1A/1B Light Chain 3B; GABARAPL1: GABA type A Receptor-Associated Protein Like 1; ATG9A: autophagy-related protein 9A; ATG13: autophagy-related protein 13; SQSTM1: Sequestosome-1/p62; WIPI2: WD repeat domain, Phosphoinositide Interacting 2; PI3P: Phosphoinositide-3-phosphate.
ABSTRACT
Repair of DNA double strand breaks (DSBs) is integral to preserving genomic integrity. Therefore, defining the mechanisms underlying DSB repair will enhance our understanding of how defects in these pathways contribute to human disease and could lead to the discovery of new approaches for therapeutic intervention. Here, we established a panel of HaloTagged DNA damage response factors in U2OS cells which enables concentration-dependent protein labeling by fluorescent HaloTag ligands. Genomic insertion of HaloTag at the endogenous loci of these repair factors preserves expression levels and proteins retain proper subcellular localization, foci-forming ability, and functionally support DSB repair. We systematically analyzed total cellular protein abundance, measured recruitment kinetics to laser-induced DNA damage sites, and defined the diffusion dynamics and chromatin binding characteristics by live-cell single-molecule imaging. Our work demonstrates that the Shieldin complex, a critical factor in end-joining, does not exist in a preassembled state and that relative accumulation of these factors at DSBs occurs with different kinetics. Additionally, live-cell single-molecule imaging revealed the constitutive interaction between MDC1 and chromatin mediated by its PST repeat domain. Altogether, our studies demonstrate the utility of single-molecule imaging to provide mechanistic insights into DNA repair, which will serve as a powerful resource for characterizing the biophysical properties of DNA repair factors in living cells.
Subject(s)
Chromatin , DNA Repair , Humans , Tumor Suppressor p53-Binding Protein 1/metabolism , DNA Breaks, Double-Stranded , DNA DamageABSTRACT
Localization of a variety of RNAs to non-membrane-bound cellular compartments such as nucleoli and Cajal bodies is critical for their stability and function. The molecular mechanisms that underly the recruitment and exclusion of RNAs from these phase-separated organelles is incompletely understood. Telomerase is a ribonucleoprotein composed of the reverse transcriptase protein telomerase reverse transcriptase (TERT), the telomerase RNA (TR), and several auxiliary proteins, including TCAB1. Here we show that in the absence of TCAB1, a large fraction of TR is tightly bound to the nucleolus, while TERT is largely excluded from the nucleolus, reducing telomerase assembly. This suggests that nuclear compartmentalization by the non-membrane-bound nucleolus counteracts telomerase assembly, and TCAB1 is required to retain TR in the nucleoplasm. Our work provides insight into the mechanism and functional consequences of RNA recruitment to organelles formed by phase separation and demonstrates that TCAB1 plays an important role in telomerase assembly.