Biology subjects

Liu, N.

Publications and source records attributed to Liu, N..

At least 181 records · Page 10Linked to original sources

bioRxiv2019

Genome assembly of the common pheasant Phasianus colchicus, a model for speciation and ecological genomics

The common pheasant (Phasianus colchicus) in the order Galliformes and the family Phasianidae, has 30 subspecies distributed across its native range in the Palearctic realm and has been introduced to Europe, North America, and Australia. It is an important game bird often subjected to wildlife management as well as a model species to study speciation, biogeography and local adaptation. However, the genomic resources for the common pheasant are generally lacking. We sequenced a male individual of the subspecies torquatus of the common pheasant with the Illumina Hiseq platform. We obtained 94.88 Gb of usable sequences by filtering out low-quality reads of the raw data generated. This resulted in a 1.02 Gb final assembly, which equals the estimated genome size. BUSCO analysis using chicken as a model showed that 93.3% of genes were complete. The contig N50 and scaffold N50 sizes were 178 kb and 10.2 Mb, respectively. All these indicate that we obtained a high-quality genome assembly. We annotated 16,485 protein-coding genes and 123.3 Mb (12.05 % of the genome) of repetitive sequences by ab initio and homology-based prediction. Furthermore, we applied a RAD-sequencing approach for another 45 individuals of seven representative subspecies in China and identified 4,376,351 novel single nucleotide polymorphism (SNPs) markers. Using this unprecedented dataset, we uncover the geographic population structure and genetic introgression among common pheasants in China. Our results provide the first high-quality reference genome for the common pheasant and a valuable genome-wide SNP database for studying population genomics and demographic history.

genomics↗

bioRxiv2019

CaMKII oxidation is a performance/disease trade-off in vertebrate evolution

Reactive oxygen species (ROS) contribute to health and disease. CaMKII is a widely expressed enzyme whose activation by oxidation of regulatory domain methionines (ox-CaMKII) contributes to cardiovascular disease, asthma, and cancer. Here we integrate comparative genomic and experimental data to show that CaMKII activation by ROS arose more than half-a-billion years ago on the vertebrate stem lineage where it constituted a bridge between ROS and increased intracellular Ca2+ release, exercise responsive gene transcription, and improved performance in skeletal muscle. These enhancements to fight-or-flight physiology were likely key in facilitating a well-evidenced shift in the behavioural ecology of our immediate chordate ancestors, and, in turn, the evolutionary success of vertebrates. Still, the ox-CaMKII innovation for augmenting performance must be considered a critical evolutionary trade-off, as it rendered us more susceptible to common and often fatal diseases linked to excessive ROS.

evolutionary biology↗

bioRxiv2019

Pumilio Proteins Exert Distinct Biological Functions and Multiple Modes of Post-Transcriptional Regulation in Embryonic Stem Cell Pluripotency and Early Embryogenesis

Gene regulation in embryonic stem cells (ESCs) has been extensively studied at the epigenetic-transcriptional levels, but not at the post-transcriptional levels. Pumilio (Pum) proteins are among the few known translational regulators required for stem cell maintenance in invertebrates and plants. Here we report the essential function of two murine Pum proteins, Pum1 and Pum2, in ESCs and early embryogenesis. Pum1/2 double mutants are developmentally delayed at the morula stage and lethal by embryonic day 8.5 (e8.5). Correspondingly, Pum1/2 double mutant ESCs display severely reduced self-renewal and differentiation, revealing the combined function of Pum1 and Pum2 in ESC pluripotency. Remarkably, Pum1-deficient ESCs show increased expression of pluripotency genes but not differentiation genes, indicating that Pum1 mainly promote differentiation; whereas Pum2-deficient ESCs show decreased expression of pluripotency genes and accelerated differentiation, indicating that Pum2 promotes self-renewal. Thus, Pum1 and Pum2 each uniquely contributes to one of the two complementary aspects of pluripotency. Furthermore, we show that Pum1 and Pum2 achieve ESC functions by forming a negative auto- and inter-regulatory feedback loop that directly regulates at least 1,486 mRNAs. Pum1 and Pum2 regulate target mRNAs not only by repressing translation as expected but also by promoting translation and enhancing or reducing mRNA stability of different target mRNAs. Together, these findings reveal the distinct roles of individual mammalian Pum proteins in ESCs and their collectively essential functions in ESC pluripotency and embryogenesis. Moreover, they demonstrate three novel modes of regulation of Pum proteins towards target mRNAs.\n\nSIGNIFICANCE STATEMENTThis report demonstrates the essential functions of mammalian Pumilio (Pum) proteins for embryonic stem cells (ESCs) pluripotency and embryogenesis. Moreover, it reveals the contrasting but complementary function of individual Pum proteins in regulating distinct aspects of ESC pluripotency, despite their largely overlapping expression and extremely high homology. Furthermore, it unravels a complex regulatory network in which Pum1 and Pum2 form a negative auto- and inter-regulatory feedback loop that regulates 1,486 mRNAs not only by translational repression as expected but also by promoting translation and enhancing or reducing stability of different target mRNAs, which reveals novel modes of post-transcriptional regulation mediated by Pum.

developmental biology↗

bioRxiv2019

Heart Rate n-Variability (HRnV) and Its Application to Risk Stratification of Chest Pain Patients in the Emergency Department

BackgroundChest pain is one of the most common complaints among patients presenting to the emergency department (ED). Causes of chest pain can be benign or life threatening, making accurate risk stratification a critical issue in the ED. In addition to the use of established clinical scores, prior studies have attempted to create predictive models with heart rate variability (HRV). In this study, we proposed heart rate n-variability (HRnV), an alternative representation of beat-to-beat variation in electrocardiogram (ECG) and investigated its association with major adverse cardiac events (MACE) for ED patients with chest pain. MethodsWe conducted a retrospective analysis of data collected from the ED of a tertiary hospital in Singapore between September 2010 and July 2015. Patients >20 years old who presented to the ED with chief complaint of chest pain were conveniently recruited. Five to six-minute single-lead ECGs, demographics, medical history, troponin, and other required variables were collected. We developed the HRnV-Calc software to calculate HRnV parameters. The primary outcome was 30-day MACE, which included all-cause death, acute myocardial infarction, and revascularization. Univariable and multivariable logistic regression analyses were conducted to investigate the association between individual risk factors and the outcome. Receiver operating characteristic (ROC) analysis was performed to compare the HRnV model (based on leave-one-out cross-validation) against other clinical scores in predicting 30-day MACE. ResultsA total of 795 patients were included in the analysis, of which 247 (31%) had MACE within 30 days. The MACE group was older and had a higher proportion of male patients. Twenty-one conventional HRV and 115 HRnV parameters were calculated. In univariable analysis, eleven HRV parameters and 48 HRnV parameters were significantly associated with 30-day MACE. The multivariable stepwise logistic regression identified 16 predictors that were strongly associated with the MACE outcome; these predictors consisted of one HRV, seven HRnV parameters, troponin, ST segment changes, and several other factors. The HRnV model outperformed several clinical scores in the ROC analysis. ConclusionsThe novel HRnV representation demonstrated its value of augmenting HRV and traditional risk factors in designing a robust risk stratification tool for patients with chest pain at the ED.

bioengineering↗

bioRxiv2019

A Clinical Phenotyping Algorithm to Identify Cases of Chronic Obstructive Pulmonary Disease in Electronic Health Records

RationaleChronic obstructive pulmonary disease (COPD) is a leading cause of mortality in the United States. Electronic health records provide large-scale healthcare data for clinical research, but have been underutilized in COPD research due to challenges identifying these individuals, especially in the absence of pulmonary function testing data.\n\nObjectivesTo develop an algorithm to electronically phenotype individuals with COPD at a large tertiary care center.\n\nMethodsWe identified individuals over 45 years of age at last clinic visit within Vanderbilt University Medical Center electronic health records. We tested phenotyping algorithms using combinations of both structured and unstructured text and examined the clinical characteristics of the resulting case sets.\n\nMeasurement and Main ResultsA simple algorithm consisting of 3 International Classification of Disease codes for COPD achieved a sensitivity of 97.6%, a specificity of 76.0%, a positive predictive value of 57.1%, and a negative predictive value of 99.0%. A more complex algorithm consisting of both billing codes and a mention of oxygen on the problem list that achieved a positive predictive value of 86.5%. However, the association of known risk factors with chronic obstructive pulmonary disease was consistent in both algorithm sets, suggesting a simple code-only algorithm may suffice for many research applications.\n\nConclusionsSimple code-only phenotyping algorithms for chronic obstructive pulmonary disease can identify case populations with epidemiologic and genetic profiles consistent with published literature. Implementation of this phenotyping algorithm will expand opportunities for clinical research and pragmatic trials for COPD.

bioinformatics↗

bioRxiv2019

Atrial fibrillation associated common risk variants in SYNE2 lead to lower expression of nesprin-2α1 and increased nuclear stiffness

RationaleAtrial fibrillation (AF) genome-wide association studies (GWAS) identified significant associations for rs1152591 and linked variants in the SYNE2 gene encoding the nesprin-2 protein that connects the nuclear membrane with the cytoskeleton\n\nObjectiveDetermine the effects of the AF-associated rs1152591 and rs1152595, two linked intronic single nucleotide polymorphisms (SNPs), on SYNE2 expression and investigate the mechanisms for their association with AF.\n\nMethods and ResultsRNA sequencing of human left atrial appendage (LAA) tissues indicated that rs1152591 and rs1152595 were significantly associated with the expressions of SYNE21, a short mRNA isoform, without an effect on the expression of the full-length SYNE2 mRNA. SYNE21 mRNA uses an alternative transcription start site and encodes an N-terminal deleted 62 kDa nesprin-21 isoform, which can act as a dominant-negative on nuclear-cytoskeleton connectivity. Western blot and qPCR assays confirmed that AF risk alleles of both SNPs were associated with lower expression of nesprin-21 in human LAA tissues. Reporter gene transfections demonstrated that the risk vs. reference alleles of rs1152591 and rs1152595 had decreased enhancer activity. SYNE2 siRNA knockdown (KD) or nesprin-21 overexpression studies in human stem cell-derived induced cardiomyocytes (iCMs) resulted in ~12.5 % increases in the nuclear area compared to controls (p<0.001). Atomic force microscopy demonstrated that SYNE2 KD or nesprin-21 overexpression led to 57.5% or 33.2% decreases, respectively, in nuclear stiffness compared to controls (p< 0.0001).\n\nConclusionsAF-associated SNPs rs1152591 and rs1152595 downregulate the expression of SYNE21, increasing nuclear-cytoskeletal connectivity and nuclear stiffness. The resulting increase in mechanical stress may play a role in the development of AF.

genomics↗

bioRxiv2019

Aspirin Compliance for Cardiovascular Disease and Colorectal Cancer Prevention in the Uninsured Population

Aspirin is an effective anti-inflammatory and antiplatelet agent as an irreversible inhibitor of cyclooxygenase. In 2016, the U.S. Preventive Services Task Force recommended aspirin for primary prevention of cardiovascular disease (CVD) and colorectal cancer (CRC) in patients aged 50 to 69 with a 10% or greater 10-year CVD risk. Due to the lack of literature describing compliance with these recommendations in the uninsured patient population, we studied the aspirin adherence for CVD and CRC prevention in several free medical clinics. We investigated the records of 8857 uninsured patients who visited nine free medical clinics in the Tampa Bay Area in 2016-2017. Aspirin compliance was assessed for patients with prior myocardial infarction (MI) or coronary artery disease (CAD). 54% (n=1467) of patients met the criteria to take aspirin for primary prevention of CVD and CRC, but just 17% of these patients aged 50-59 were on the medication. 16% percent of patients aged 60-69 were taking aspirin and significantly more men than women were on aspirin (p=0.025). Of the 306 patients who had prior MI or CAD, 50% were on the medication for secondary prevention. Among the uninsured population, there is low compliance with recommendations for aspirin usage to reduce the risk of CVD and CRC. This study demonstrates that further improvements are needed to increase adherence to current guidelines and address barriers uninsured patients may face in maintaining their cardiovascular and colorectal health.

epidemiology↗

bioRxiv2019

Contrasting biogeographic patterns of bacterial and archaeal diversity in the top- and subsoils of temperate grasslands

Biogeographic patterns and drivers of soil microbial diversity have been extensively studied in the past few decades. However, most research has focused on the topsoil while the subsoil is assumed to have similar microbial diversity patterns as the topsoil. Here we compare patterns and drivers of microbial diversity in the top- (0-10 cm) versus subsoils (30-50 cm) of temperate grasslands in Inner Mongolia of China along an aridity gradient covering a ~1500-km transect from arid to mesic ecosystems. Counter to the conventional assumption, we find contrasting biogeographic patterns of diversity and influencing factors for different bacterial and archaeal groups and between depths. While bacterial diversity increases with increasing aridity, archaeal diversity decreases. Microbial diversity in the topsoil is most strongly influenced by aboveground vegetation, but is most strongly influenced by historical temperature anomaly since the Last Glacial Maximum (LGM) in the subsoil. Moreover, the biogeographic patterns of top-subsoil diversity difference varies for different microbial groups and is overall most strongly influenced by soil fertility difference between depths and historical temperature anomaly. These findings suggest that diversity patterns observed in the topsoil may not be readily applied to the subsoil horizons. For the subsoil in particular, historical climate plays a vital role in the distribution of various microbial groups. Overall, our study provides novel information for understanding and predicting soil microbial diversity patterns at depth.\n\nIMPORTANCEExploring the biogeographic patterns of soil microbial diversity is critical for understanding mechanisms underlying the response of soil processes to climate change. Using top- and subsoils from a ~1500-km temperature grassland transect, we find divergent patterns of microbial diversity and its determinants in the top-versus subsoils. Furthermore, we find important legacy effect of historical climate change on the microbial diversity of subsoil but not topsoil. Our findings challenge the conventional assumption of similar geographic patterns of soil microbial diversity along soil profiles and help to improve our understanding of how soil microbial communities may respond to future climate change in different regions with varied climate history.

microbiology↗

bioRxiv2019

Methods for the generation of heritable germline mutations in the disease vector Culex quinquefasciatus using CRISPR/Cas9.

Culex quinquefasciatus is vector of many diseases that adversely impact human and animal health; however, compared to other mosquito vectors limited genome engineering technologies have been characterized for this vector. CRISPR-Cas9 based technologies are a powerful tool for genome engineering and functional genomics and consequently have transformed genomics studies in many organisms. Our objective was to improve upon the limited technologies available for genome editing in Cx. quinquefasciatus to create a reproducible and straightforward method for CRISPR-Cas9-targeted mutagenesis in this vector. Here we describe methods to both improve embryo survival rates as well as mutagenesis rates by optimizing injection supplies and equipment, embryo injection procedures, embryo handling and gRNA target design. Through these efforts, we achieved embryo survival rates and germline mutagenesis rates that greatly exceed any previously reported rates in this vector. This work was also the first characterize the white gene marker, which is a valuable phenotypic marker for future transgenesis or mutagenesis of this vector. In the end, these tools provide the framework for future functional genomic studies in this important disease vector and may support the development of future gene drive and genetic technologies that can be used to control this vector.

bioengineering↗

bioRxiv2019

TRIM21-SERPINB5 aids GMPS repression to protect nasopharyngeal carcinoma cells from radiation-induced apoptosis

Nasopharyngeal carcinoma (NPC) is the most prevalent head and neck malignancy in South China and Southeast Asia. The main NPC treatment strategy is radiotherapy. However, recurrence resulting from radioresistance is a leading clinical bottleneck. Revealing the mechanism of NPC radioresistance would help improve the therapeutic effect. Here, our work reveals that TRIM21 (tripartite motif-containing 21) functions as an oncogene in NPC progression, and its ablation increases NPC cell radiosensitivity. Further analysis indicated that TRIM21 represses TP53 expression by mediating GMPS (guanine monophosphate synthase) ubiquitination and degradation after ionizing radiation. Mass spectrometry and co-immunoprecipitation showed that SERPINB5 (serpin family B member 5) interacts with both TRIM21 and GMPS. Epistatic analysis showed that SERPINB5 acts as an adaptor to recruit GMPS and introduce TRIM21 for ubiquitination. The in vitro and in vivo results validated the finding that SERPINB5 promotes NPC cell radioresistance. Furthermore, immunohistochemistry indicated that radioresistant patients have higher SERPINB5 expression. Overall, our data show that TRIM21-SERPINB5-mediated GMPS degradation facilitates TP53 repression, which promotes the radioresistance of NPC cells.

cancer biology↗

bioRxiv2019

VRmol: an Integrative Cloud-Based Virtual Reality System to Explore Macromolecular Structure

Past decades have seen much effort devoted to the tool development for biomacromolecule structural visualization and analysis. In recent years, we have witnessed tremendous advancement in computational technologies, especially in graphics and internet. Here we present an integrative cloud-based, virtual reality (VR) system -- VRmol that exploits these technology advances to visualize and study macromolecule structures in various forms. The immersive environment can improve user experiences in visualization, calculation, and editing of complex structures with more intuitive perception. VRmol is also a system with cloud-based capability to directly access disease-related databases, and couples the structure with genomic variation as well corresponding drugs information in a visual interface, thus serving as an integrative platform to aid structure-based translational research and drug design. VRmol is freely available at https://VRmol.net.

bioinformatics↗

bioRxiv2019

The vagino-cervical microbiome as a woman's life history

The gut microbiome has been the center of attention for human commensal microbiome studies. The vaginal microbiome is also densely populated with bacteria, viruses and fungi, and the presence of microorganisms beyond the cervix is increasingly reported in non-infectious conditions1-3. Due to the over 90% of human sequences in female reproductive tract samples3,4, metagenomic information has been very limited. 16S rRNA gene amplicon sequencing studies have identified community types in the vaginal microbiota, and observed its dynamic changes due to menstrual cycles and sexual behaviors in small cohorts5,6. Here we perform metagenomic shotgun sequencing on cervical samples from 516 women of reproductive age (more than 10-fold of the Human Microbiome Project (HMP)4), and dissect major factors, especially pregnancy and delivery histories and contraception methods on the microbiome composition. Features of other body sites, such as mood fluctuations and facial speckles could potentially be deduced from the vagino-cervical microbiome. Our results offer an unprecedented glimpse into the microbiota in the female reproductive tract and imply disease susceptibilities that may be relieved by behavioural changes.

microbiology↗

bioRxiv2019

Heterologous expression of Stlac2, a laccase isozyme of Setosphearia turcica, and the ability of decolorization of malachite green

Laccases can catalyze monoelectronic oxidation and have shown to have an increasing value in industrial application. In this study, as identified by Native-PAGE and ESI-MS/MS, ascomycetous fungus Setosphaeria turcica produced three laccase isozymes: Stlac1, Stlac2, and Stlac6. Stlac2 was heterologously expressed in both eukaryotic and prokaryotic expression systems. The eukaryotic recombinant Stlac2 expressed in Pichia pastoris was inactive, and also showed a higher molecular weight than predicted because of glycosylation. The depression of laccase activity was attributable to the incorrect glycosylation at Asn97. Stlac2 expressed in Escherichia coli and after being renaturated from the inclusion body, the recombinant Stlac2 exhibited activity of 28.23 U/mg with 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. The highest activity was observed at pH of 4.5 and the temperature of 60 {degrees}C. The activity of recombinant Stlac2 was inhibited by 10 mM Na+, Mg2+, Ca2+, Mn2+, and increased by 10 mM of Fe3+ with a relatively activity of 315% compared with no addition. Cu2+ did not affect enzyme activity. Recombinant Stlac2 was capable of decolorizing 67.08% of 20 mg/L malachite green in 15 min without any mediators. It is suggested that Stlac2 has potential industrial applications. ImportanceSetosphaeria turcica, an ascomycetous fungus causes northern corn leaf blight, product three laccase isozymes identified by Native-PAGE and ESI-MS/MS. The major expression laccase gene StLAC2 was expression in both eukaryotic and prokaryotic expression systems, which found incorrect glycosylation at Asn97 may result in the depression of laccase activity. The heterologous laccase Stlac2 decolorize organic dye malachite green, which had a potential industrial application.

bioengineering↗

bioRxiv2019

CUT&RUNTools: a flexible pipeline for CUT&RUN processing and footprint analysis

We introduce CUT&RUNTools as a flexible, general pipeline for facilitating the identification of chromatin-associated protein binding and genomic footprinting analysis from antibody-targeted CUT&RUN primary cleavage data. CUT&RUNTools extracts endonuclease cut site information from sequences of short read fragments and produces single-locus binding estimates, aggregate motif footprints, and informative visualizations to support the high-resolution mapping capability of CUT&RUN. CUT&RUNTools is available at https://bitbucket.org/qzhudfci/cutruntools/.

genomics↗

bioRxiv2018

Makorin1 controls embryonic patterning by alleviating Bruno-mediated repression of oskar translation

Makorins are evolutionary conserved proteins that contain C3H-type zinc finger modules and a RING E3 ubiquitin ligase domain. In Drosophila maternal Makorin 1 (Mkrn1) has been linked to embryonic patterning and germ cell specification. Here, we show that Mkrn1 is required for translational activation of oskar, whose product is critical for axis specification and germ plasm assembly. We demonstrate that Mkrn1 interacts with poly(A) binding protein (pAbp) and binds osk 3 UTR in a region adjacent to A-rich sequences. This binding site also overlaps with Bruno (Bru) responsive elements (BREs), which regulate osk translation. We observe increased association of the translational repressor Bru with osk mRNA upon depletion of Mkrn1, implying that the two proteins compete for osk binding. Consistently, reducing Bru dosage partially rescues viability and Osk protein level in ovaries from Mkrn1 females. We conclude that Mkrn1 controls embryonic patterning and germ cell formation by specifically activating osk translation via displacing Bru from its 3 UTR.

developmental biology↗

bioRxiv2018

Oxytocin involves in chronic stress-evoked melanoma metastasis via β-arrestin 2 mediated-pathways

Stress is associated with an increased risk of lung metastasis in melanoma. However, the underlying mechanism is elusive. Oxytocin (OXT), a neurohormone produced by the hypothalamus, plays a vital role in laboring induction and lactation. Emerging evidence suggests that OXT also regulates human emotions, social cognition, social behaviors and stress-related disorders. Here, we reported that a significant up-regulation of oxytocin receptors (OXTRs) was observed in malignant melanoma. The activation of oxytocin receptors (OXTRs) dramatically promoted migration, invasion and angiogenesis but not the proliferation of melanoma cells in vitro and in vivo via {beta}-arrestin 2-dependent ERK-VEGF/ MMP-2 pathway. Next, chronic restraint stress significantly elevated the plasma level of OXT. Notably, 21 days chronic restraint stress facilitated lung metastasis of melanoma and reduced overall survival in mice, which were largely abrogated by knocking down either OXTR or {beta}-arrestin 2. These findings provide evidence that chronic stress hormone-OXT promotes lung metastasis of melanoma via a {beta}-arrestin 2-dependent mechanism and suggest that OXT, a novel pro-metastasis factor, is a potential therapeutic target for melanoma.\n\nImportance of the StudyOxytocin, a neuropeptide produced in the hypothalamus that is involved in lactation and parturition, play a vital role in chronic stress-evoked melanoma metastasis via {beta}-arrestin 2-mediated ERK signaling pathway, which provides a potential therapeutic target for melanoma.

cancer biology↗

bioRxiv2018

A VTA GABAergic neural circuit mediates visually evoked innate defensive responses

Innate defensive responses are essential for animal survival and are conserved across species. The ventral tegmental area (VTA) plays important roles in learned appetitive and aversive behaviors, but whether it plays a role in mediating or modulating innate defensive responses is currently unknown. We report that GABAergic neurons in the mouse VTA (VTAGABA+) are preferentially activated compared to VTA dopaminergic (VTADA+) neurons when a threatening visual stimulus evokes innate defensive behavior. Functional manipulation of these neurons showed that activation of VTAGABA+ neurons is indispensable for looming-evoked defensive flight behavior and photoactivation of these neurons is sufficient for looming-evoked defensive-like flight behavior, whereas no such role can be attributed for VTADA+ neurons. Viral tracing and in vivo and in vitro electrophysiological recordings showed that VTAGABA+ neurons receive direct excitatory inputs from the superior colliculus (SC). Furthermore, we showed that glutamatergic SC-VTA projections synapse onto VTAGABA+ neurons that project to the central nucleus of the amygdala (CeA) and that the CeA is involved in mediating the defensive behavior. Our findings demonstrate that visual information about aerial threats access to the VTAGABA+ neurons mediating innate behavioral responses, suggesting a more general role for the VTA.

neuroscience↗

bioRxiv2018

Loss of Rap1 supports recombination-based telomere maintenance independent of RNA-DNA hybrids in fission yeast

A subset of cancers rely on telomerase-independent mechanisms to maintain their chromosome ends. The predominant \"alternative lengthening of telomeres\" pathway appears dependent on homology-directed repair (HDR) to maintain telomeric DNA. However, the molecular changes needed for cells to productively engage in telomeric HDR are poorly understood. To gain new insights into this transition, we monitored the state of telomeres during serial culture of fission yeast (Schizosaccharomyces pombe) lacking the telomerase recruitment factor Ccq1. Rad52 is loaded onto critically short telomeres shortly after germination despite continued telomere erosion, suggesting that recruitment of recombination factors is not sufficient to maintain telomeres in the absence of telomerase function. Instead, survivor formation coincides with the derepression of telomeric repeat-containing RNA (TERRA). In this context, degradation of TERRA associated with the telomere in the form of R-loops drives a severe growth crisis, ultimately leading to a novel type of survivor with linear chromosomes and altered cytological telomere characteristics, including the loss of the shelterin component Rap1 (but not the TRF1/TRF2 orthologue, Taz1) from the telomere. We demonstrate that deletion of Rap1 is protective in this context, preventing the growth crisis that is otherwise triggered by degradation of telomeric R-loops in survivors with linear chromosomes. These findings suggest that up-regulation of telomere-engaged TERRA or altered recruitment of shelterin components can support telomerase-independent telomere maintenance.

genetics↗