Plasminogen activator urokinase interacts with the fusion protein and antagonizes the growth of Peste des petits ruminants virus.

Peste des petits ruminants is an acute and highly contagious disease caused by the Peste des petits ruminants virus (PPRV). Host proteins play a crucial role in viral replication. However, the effect of fusion (F) protein-interacting partners on PPRV infection is poorly understood. In this study, we found that the expression of goat plasminogen activator urokinase (PLAU) gradually decreased in a time- and dose-dependent manner in PPRV-infected goat alveolar macrophages (GAMs). Goat PLAU was subsequently identified using co-immunoprecipitation and confocal microscopy as an F protein binding partner. The overexpression of goat PLAU inhibited PPRV growth and replication, whereas silencing goat PLAU promoted viral growth and replication. Additionally, we confirmed that goat PLAU interacted with a virus-induced signaling adapter (VISA) to antagonize F-mediated VISA degradation, increasing the production of type I interferon. We also found that goat PLAU reduced the inhibition of PPRV replication in VISA-knockdown GAMs. Our results show that the host protein PLAU inhibits the growth and replication of PPRV by VISA-triggering RIG-I-like receptors and provides insight into the host protein that antagonizes PPRV immunosuppression.IMPORTANCEThe role of host proteins that interact with Peste des petits ruminants virus (PPRV) fusion (F) protein in PPRV replication is poorly understood. This study confirmed that goat plasminogen activator urokinase (PLAU) interacts with the PPRV F protein. We further discovered that goat PLAU inhibited PPRV replication by enhancing virus-induced signaling adapter (VISA) expression and reducing the ability of the F protein to degrade VISA. These findings offer insights into host resistance to viral invasion and suggest new strategies and directions for developing PPR vaccines.

Prediction of local tumor progression after microwave ablation for early-stage hepatocellular carcinoma with machine learning.

Objectives: Local tumor progression (LTP) is a major constraint for achieving technical success in microwave ablation (MWA) for the treatment of early-stage hepatocellular carcinoma (EHCC). This study aims to develop machine learning (ML)-based predictive models for LTP after initial MWA in EHCC. Materials and Methods: A total of 607 treatment-naïve EHCC patients (mean ± standard deviation [SD] age, 57.4 ± 10.8 years) with 934 tumors according to the Milan criteria who subsequently underwent MWA between August 2009 and January 2016 were enrolled. During the same period, 299 patients were assigned to the external validation datasets. To identify risk factors of LTP after MWA, clinicopathological data and ablation parameters were collected. Predictive models were developed according to 21 variables using four ML algorithms and evaluated based on the area under the receiver operating characteristic curve (AUC) with 95% confidence intervals (CIs). Results: After a median follow-up time of 28.7 months (range, 7.6-110.5 months), 6.9% (42/607) of patients had confirmed LTP in the training dataset. The tumor size and number were significantly related to LTP. The AUCs of the four models ranged from 0.791 to 0.898. The best performance (AUC: 0.898, 95% CI: [0.842 0.954]; SD: 0.028) occurred when nine variables were introduced to the CatBoost algorithm. According to the feature selection algorithms, the top six predictors were tumor number, albumin and alpha-fetoprotein, tumor size, age, and international normalized ratio. Conclusions: Out of the four ML models, the CatBoost model performed best, and reasonable and precise ablation protocols will significantly reduce LTP.

How effective are community-based disaster reduction strategies? Evidence from the largest-scale program so far.

Strategies of community-based disaster risk reduction have been advocated for more than 2 decades. However, we still lack in-depth quantitative assessments of the effectiveness of such strategies. Our research is based on a national experiment in this domain: the "Comprehensive Disaster Reduction Demonstration Community" project, a governmental program running in China since 2007. Information on more than 11,000 demonstration communities was collected. Combined with the local disaster information and socioeconomic conditions, the spatiotemporal characteristics of these communities over 12 years and their differences in performance by region and income group were analyzed. We performed an attribution analysis for disaster risk reduction effectiveness. This is the first time a series of quantitative evaluation methods have been applied to verify the effectiveness of a large-scale community-based disaster risk reduction project, both from the perspective of demonstrative effects and loss reduction benefits. Here, we find that the project is obviously effective from these two perspectives, and the disaster loss reduction effectiveness illustrates clear regional differences, where the regional economic level and hazard severity act as important drivers. Significant differences of urban-rural and income call for matching fortification measures, and the dynamic management of demonstration community size is required, since the loss reduction benefit converges when the penetration rate of the demonstration community reaches approximately 4% in a province. These and further results provide diverse implications for community-based disaster risk reduction policies and practices.

Effects of physical training on cyclists' speed / Efeitos do treinamento físico sobre a velocidade dos ciclistas / Efectos del entrenamiento físico en la velocidad de los ciclistas

ABSTRACT Introduction Cycling is a competitive sport and has high demands on the athletes themselves, such as versatile use of skills, physical fitness, and explosiveness. Objective Increase the specific speed endurance of athletes. Methods Fourteen prominent male short-distance cyclists were divided into a group of constant cyclists (CG group, n=7) and a group of decreasing cyclists (DG group, n=7) and subjected to a physical training protocol. Results Peak activity and average endurance improved in both groups of athletes; rest periods improved significantly compared to the previous ones, and deceleration decreased by 12.2% and 14.6% in the CG and DG groups, respectively, immediately after the end of the rest period. Latency decreased significantly in both groups (P<0.05). Conclusion Compared with the program with the same number of rides, the training load in the stage-based pilot program with the decreasing number of rides was higher, and the training effect was more significant in the improvement of peak power, 1 km total power maintenance capacity, and anaerobic threshold. The energy supply ratio of aerobic metabolism during exercise at the above intensity was also improved. Level of evidence II; Therapeutic studies - investigation of treatment outcomes.

RESUMO Introdução O ciclismo é um esporte de competição e tem altas exigências para os próprios atletas, tais como o uso versátil de habilidades, aptidão física e explosividade. Objetivo Aumentar a resistência de velocidade específica dos atletas. Métodos Catorze proeminentes ciclistas masculinos de curta distância foram divididos em um grupo de número constante de ciclistas (grupo CG, n=7) e um grupo de número decrescente de ciclistas (grupo DG, n=7), submetidos a um protocolo de treinamento físico. Resultados Os picos de atividade e média de resistência sofrem uma melhoria nos dois grupos de atletas, períodos de repouso melhoraram significativamente em comparação com os anteriores, a desaceleração diminuiu 12,2% e 14,6% nos grupos CG e DG, respectivamente, imediatamente após o final do período de repouso. A latência diminuiu significativamente em ambos os grupos (P<0,05). Conclusão Em comparação com o programa com o mesmo número de pedaladas, a carga de treinamento no estágio baseado no programa piloto com o número decrescente de pedaladas foi maior, o efeito do treinamento foi mais significativo na melhoria da potência de pico, na capacidade de manutenção de potência de 1 km total e no limiar anaeróbico. A relação do fornecimento de energia do metabolismo aeróbico durante o exercício com intensidade acima da intensidade também foi aprimorada. Nível de evidência II; Estudos terapêuticos - investigação dos resultados do tratamento.

RESUMEN Introducción El ciclismo es un deporte de competición y tiene grandes exigencias para los propios atletas, como el uso versátil de las habilidades, la aptitud física y la explosividad. Objetivo Aumentar la resistencia a la velocidad específica de los atletas. Métodos Catorce destacados ciclistas masculinos de corta distancia fueron divididos en un grupo de número constante de ciclistas (grupo CG, n=7) y un grupo de número decreciente de ciclistas (grupo DG, n=7), sometidos a un protocolo de entrenamiento físico. Resultados La actividad máxima y la resistencia media experimentan una mejora en ambos grupos de atletas, los periodos de descanso mejoran significativamente en comparación con los anteriores, la desaceleración disminuye un 12,2% y un 14,6% en los grupos CG y DG, respectivamente, inmediatamente después de finalizar el periodo de descanso. La latencia disminuyó significativamente en ambos grupos (P<0,05). Conclusión En comparación con el programa con el mismo número de salidas, la carga de entrenamiento en el programa piloto basado en etapas con el número de salidas decreciente fue mayor, el efecto del entrenamiento fue más significativo en la mejora de la potencia máxima, la capacidad de mantenimiento de la potencia total en 1 km y el umbral anaeróbico. También mejoró la relación de suministro de energía del metabolismo aeróbico durante el ejercicio a una intensidad superior. Nivel de evidencia II; Estudios terapéuticos - investigación de los resultados del tratamiento.

Genotype Complements the Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by Nanopore Long-Read Sequencing in a Large DCM Family.

Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20−40% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the causal variant in a multi-generational pedigree of DCM. A four-generation family with familial DCM was investigated. Next-generation sequencing (NGS) was performed on 22 family members. Skin biopsies from two affected family members were used to generate iPSCs, which were then differentiated into iPSC-CMs. Short-read RNA sequencing was used for the evaluation of the target gene expression, and long-read RNA nanopore sequencing was used to evaluate the relevance of the splice variants. The pedigree suggested a highly penetrant, autosomal dominant mode of inheritance. The phenotype of the family was suggestive of laminopathy, but previous genetic testing using both Sanger and panel sequencing only yielded conflicting evidence for LMNA p.R644C (rs142000963), which was not fully segregated. By re-sequencing four additional affected family members, further non-coding LMNA variants could be detected: rs149339264, rs199686967, rs201379016, and rs794728589. To explore the roles of these variants, iPSC-CMs were generated. RNA sequencing showed the LMNA expression levels to be significantly lower in the iPSC-CMs of the LMNA variant carriers. We demonstrated a dysregulated sarcomeric structure and altered calcium homeostasis in the iPSC-CMs of the LMNA variant carriers. Using targeted nanopore long-read sequencing, we revealed the biological significance of the variant c.356+1G>A, which generates a novel 5' splice site in exon 1 of the cardiac isomer of LMNA, causing a nonsense mRNA product with almost complete RNA decay and haploinsufficiency. Using novel molecular analysis and nanopore technology, we demonstrated the pathogenesis of the rs794728589 (c.356+1G>A) splice variant in LMNA. This study highlights the importance of precise diagnostics in the clinical management and workup of cardiomyopathies.

Projections of land use change and habitat quality assessment by coupling climate change and development patterns.

Exploring future land use changes and assessing the habitat quality remains a challenging topic for watershed ecological sustainability. However, most studies ignore the effects of coupled climate change and development patterns. In this study, a framework for assessing habitat quality under the influence of future land use change is constructed based on exploring the driving forces of land use change factors and integrating the system dynamics (SD) model, future land use simulation (FLUS) model and InVest model. The framework enables the projection of land use change and the assessment of habitat quality in the context of future climate change and different development strategies. Applying the framework to the Weihe River Basin, the main driving forces of land-use change in the Weihe River Basin were identified based on geographical detectors, and habitat quality assessment was realized for the Weihe River Basin under the coupled scenarios of three typical shared socioeconomic pathways and future development patterns (SSP126-EP, SSP245-ND, SSP585-EG). The results show that 1) population, precipitation, and temperature are the major driving factors for land use change. 2) The coupling model of SD and FLUS can effectively simulate the future trend of land use change, the relative error is within 2 %, and the overall accuracy is 93.58 %. 3) Significant differences in habitat quality as a result of modifications in land use patterns in different contexts. Affected by ecological protection, the habitat quality in SSP126-EP was significantly better than that in SSP245-ND and SSP585-EG. This research can provide references for future watershed ecological management decisions.

Single-molecule, full-length transcript isoform sequencing reveals disease-associated RNA isoforms in cardiomyocytes.

Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we establish an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generate a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms ( http://steinmetzlab.embl.de/iBrowser/ ). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identify 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by the discovery of IMMT isoforms mis-spliced by RBM20 mutations. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms, providing more immediate biological interpretation and higher resolution transcriptome comparisons.

NEOGAMES: A Serious Computer Game That Improves Long-Term Knowledge Retention of Neonatal Resuscitation in Undergraduate Medical Students.

Background: Serious games are potential alternatives for supplementing traditional simulation-based education for neonatal resuscitation training. However, evidence regarding the benefits of using serious games to improve long-term knowledge retention of neonatal resuscitation in undergraduate medical students is lacking. Objective: We designed a serious computer game "NEOGAMES" to train undergraduate medical students in neonatal resuscitation in a cost-friendly and accessible way and to examine whether serious game-based training improves long-term knowledge retention in medical students. Methods: "NEOGAMES" consists of a screen with images of an incubator, a baby, visual objects, anatomy, action cards, monitors, real-time feedback, and emotional components. Undergraduate medical students from Shanghai Medical College of Fudan University were invited to participate and were allocated to a game group or a control group. Participants in the game group played the game before the training. All the participants completed three written tests, pre- and post-training knowledge tests and a follow-up test after 6 months. Results: Eighty-one medical students participated in the study. The student demographic characteristics of the groups were comparable, including sex, age, and grade point average (GPA). Significant short-term knowledge improvement was noticed only for male students in the game group based on their 5.2-point higher test scores than those of the controls (p = 0.006). However, long-term knowledge improvement at 6 months was identified for both male and female students in the game group, with test scores 21.8 and 20 points higher, respectively, than those of the controls (P < 0.001). The long-term knowledge retention in the game group was almost 3 times higher than that in the control group. Conclusions: Long-term knowledge retention was nearly 3 times higher for the game group than for the control group. The improvement in knowledge supports the use of serious games for undergraduate medical education.

Preparation of loratadine nanocrystal tablets to improve the solubility and dissolution for enhanced oral bioavailability.

OBJECTIVES: Loratadine is a selective H1 receptor inhibitor that has been widely used in the clinical treatment of allergic diseases. Here we aimed to develop a novel solid loratadine nanocrystal to increase the low and pH-dependent water solubility for bioavailability enhancement. METHODS: Loratadine solid nanocrystal was developed through high-speed shear-high pressure homogenization followed by freeze-drying, which was further prepared into tablets through direct compression. The formulation and process parameter were screened. Furthermore, the characterization and oral bioavailability of loratadine nanocrystal were studied. KEY FINDINGS: The loratadine nanocrystal had the satisfactory particle size of 425.9 nm and great redispersibility, which was mainly attributed to the addition of Pluronic F127 and polyvinylpyrrolidone K17 as the stabilizer. The saturation solubility of the loratadine nanocrystal was increased to 3.81, 3.22 and 2.57-fold that of the crude drug in water, pH 6.8 and pH 4.5 buffer respectively. Furthermore, the pharmacokinetic studies in rats revealed that the AUC (0-∞) of the nanocrystal tablets was 2.38-fold that of raw tablets and 1.94-fold that of commercial tablets, respectively. CONCLUSIONS: The nanocrystal tablets could significantly improve the oral bioavailability of loratadine, which would also be a promising approach to enhance the solubility of insoluble drugs.

Regulation of secondary metabolite biosynthesis in Monascus purpureus via cofactor metabolic engineering strategies.

This study investigated the effects of cofactor metabolism on secondary metabolite production in M. purpureus through the application of different cofactor engineering strategies. Total pigment production dramatically increased by 39.08% and 40.89%, and yellow pigment production increased by 74.62% and 114.06% after the addition of 1.0 mg/L of the exogenous cofactor reagents methyl viologen and rotenone, respectively, in submerged batch-fermentation. The extracellular red pigment tone changed to yellow with the application of electrolytic stimulation at 800 mV/cm2, but almost no citrinin production was detected. In addition, the total pigment, yellow pigment and citrinin production increased by 35.46%, 54.89% and 6.27% after disruption of the nuoⅠ gene that encodes NADH-quinone oxidoreductase, respectively. Thus, cofactor metabolic engineering strategies could be extended to the industrial production of Monascus pigment or high yellow pigment with free citrinin production.

iPSC Modeling of RBM20-Deficient DCM Identifies Upregulation of RBM20 as a Therapeutic Strategy.

Recent advances in induced pluripotent stem cell (iPSC) technology and directed differentiation of iPSCs into cardiomyocytes (iPSC-CMs) make it possible to model genetic heart disease in vitro. We apply CRISPR/Cas9 genome editing technology to introduce three RBM20 mutations in iPSCs and differentiate them into iPSC-CMs to establish an in vitro model of RBM20 mutant dilated cardiomyopathy (DCM). In iPSC-CMs harboring a known causal RBM20 variant, the splicing of RBM20 target genes, calcium handling, and contractility are impaired consistent with the disease manifestation in patients. A variant (Pro633Leu) identified by exome sequencing of patient genomes displays the same disease phenotypes, thus establishing this variant as disease causing. We find that all-trans retinoic acid upregulates RBM20 expression and reverts the splicing, calcium handling, and contractility defects in iPSC-CMs with different causal RBM20 mutations. These results suggest that pharmacological upregulation of RBM20 expression is a promising therapeutic strategy for DCM patients with a heterozygous mutation in RBM20.

Positive impact of COVID-19 on career choice in pediatric medical students: a longitudinal study.

BACKGROUND: On March 11th, 2020, the WHO made the assessment that coronavirus disease 2019 (COVID-19) could be characterized as a pandemic. Medical students experienced a greater degree of anxiety and psychological stress than during previous pandemics. Negative emotions were related to decreased medical career interest, increased career choice regret and dropout rates in medical students, which affected academic and professional development. The goal of this study was to investigate the impact of the current COVID-19 outbreak on the career preferences of pediatric medical students and to explore the underlying factors contributing to it. METHODS: A prospective, longitudinal study was conducted among all 120 pediatric medical students from Shanghai Medical College of Fudan University on November 23rd, 2019, and February 21st, 2020 using a 7-item online questionnaire about career choice. RESULTS: A total of 106 (41 male and 65 female) students with a mean age of 21 years consented to participate in this study. The response rate was 100% in November 2019 and 98.1% in February 2020. Since the outbreak, career choices to practice medicine or pediatrics did not drop significantly, decreasing by only 4.3% and 2.2%, respectively. There was a positive impact of the COVID-19 outbreak on strengthening 66.7% of students' beliefs and choices to become good pediatricians (P<0.001). Only 14 students (13.5%) thought that COVID-19 had a negative impact on their career choices, but the majority of them were in the 3rd (28.6%) and 4th (64.3%) years of medical education and had insufficient knowledge about the hospital environment and clinics. CONCLUSIONS: The outbreak of COVID-19 might have an overall positive impact on career choice by strengthening students' belief and choice to become good doctors and may decrease the choice regret and drop rates of the next generation of doctors. Special attention should be paid to students with insufficient clinical experience. Good protection for students, sharing outstanding stories regarding fighting the pandemic, and innovations of needs-based curriculum could be helpful during this pandemic. Future studies are warranted to confirm these findings.

Effects of pigment and citrinin biosynthesis on the metabolism and morphology of Monascus purpureus in submerged fermentation.

The effects of the secondary metabolite biosynthesis on the metabolism and morphology of the Monascus purpureus were investigated in this study. Hypha and septum length became longer after deletion of genes pigR and pksCT in M. purpureus LQ-6 by Agrobacterium tumefaciens-mediated transformation technology, highly branched hyphae, much smaller and freely dispersed mycelial pellets were observed in M. purpureus. Compared with that in the wild-type, the level of intracellular NADH and NADPH was almost constant in M. purpureus ΔpigR at 4 days, but the NADH and NADPH levels decreased by 1.58-fold and 3.71-fold in M. purpureus ΔpksCT. The present study can not only provide a kind of strategy to improve the Monascus pigments production, but also provide theoretical support for the further study of relationship between the secondary metabolites, metabolism and morphological change.

Performance of heat-health warning systems in Shanghai evaluated by using local heat-related illness data.

In response to more frequent heatwaves, various regional or national heat-health warning systems (HHWSs) have been developed recently as adaptation measures. A wide range of methodologies have been utilized to issue warnings, as there is no universal definition of "heat event" or "heatwave", nor are there quantified thresholds of human-health tolerance to extreme weather. The performance of these warning systems has rarely been evaluated with actual heat-health data, especially the morbidity data, in regions with severe impact. In this study, we assessed the performance of the Shanghai HHWS based on heat-related illness data collected by the Chinese Center for Disease Control and Prevention (China CDC) and then conducted a comparative analysis among the Shanghai HHWS, the China Meteorological Administration HHWS, the Chinese national standard for heatwave indexes, the heat index adopted by the USA's National Weather Service and the definition suggested by the World Meteorological Organization to understand their potential performance for application in Shanghai and to evaluate the temperature thresholds and different meteorological indices employed. The results show that: 1) during the research period, 50% of heat-related illnesses and 58.2% of heat-related deaths in Shanghai occurred on dates that had no heat warnings; 2) for the current threshold (35 °C), the single metric of temperature outperformed the temperature-duration two-parameter method; 3) different from existing studies, while infants and seniors are deemed as vulnerable population groups to heat, young and middle-aged males were found to suffer more heat-related illnesses in hot weather. More detailed analyses reveal that the performance of heat-health warning systems needs to be evaluated and revised periodically, and warning thresholds utilized must be localized to reflect public tolerance to heat and to address the vulnerability of target population groups. Temperature is the dominant threshold in heat-related morbidity and mortality analysis. While a decrease in the temperature threshold would definitely increase the warning frequency and socioeconomic costs, it might also cause warning fatigue. The trade-off between these two aspects is essential for decision-makers and other stakeholders in HHWS design and improvement.

Disruption of the Ergosterol Biosynthetic Pathway Results in Increased Membrane Permeability, Causing Overproduction and Secretion of Extracellular Monascus Pigments in Submerged Fermentation.

Because Monascus pigments (MPs) predominantly accumulate in the cytoplasm during submerged fermentation, many biotechnologies are applied to enhance the production of extracellular MPs (exMPs) to reduce the downstream processing costs. In this study, the genes monascus_7017 and monascus_8018, identified as ERG4 genes, were knocked out to disrupt the ergosterol biosynthetic pathway and enhance the production of exMPs in Monascus purpureus LQ-6. Double-deletion of EGR4 in M. purpureus LQ-6 reduced ergosterol concentration by 57.14% and enhanced exMP production 2.06-fold. In addition, integrated transcriptomic and proteomic analyses were performed to elucidate the transmembrane secretion mechanism of exMPs based on the relationship between ergosterol synthesis and membrane permeability, which revealed that several metabolic pathways were noticeably dynamic, including fatty acid degradation, amino acid metabolism, energy metabolism, carbohydrate metabolism, and transport. These findings therefore clarified the secretion mechanism of exMPs and provide a novel strategy for further enhancement of exMP production in submerged fermentation.

Enhancement of Monascus pigment productivity via a simultaneous fermentation process and separation system using immobilized-cell fermentation.

A mutant Monascus purpureus strain, M183, which produced monascus pigments (MPs) at 8460 U/g via solid-state batch-fermentation, was generated using the atmospheric and room temperature plasma (ARTP) mutation system. The optimal glucose concentration (80 g/L) in traditional fermentation media that yielded the highest MPs productivity was determined. Response surface methodology (RSM) was applied to maximize MPs production using liquid-state batch-fermentation. Under optimal conditions (0.58 g/L MgSO4·7H2O, 0.02 g/L ZnSO4·7H2O, 0.02 g/L FeSO4·7H2O and 4.85 g/L NH4NO3), 207.67 U/mL of MPs were produced with 98.12% validity based on the predicted value. Extracellular MPs production increased significantly to 35.52 U/mL, compared to 14.19 U/mL of the original strain, M. purpureus LQ-6. M. purpureus spores immobilized in sodium alginate were studied. A simultaneous fermentation and separation system was established for MPs using the novel absorption resin LX300C to enhance production efficiency of extracellular MPs.

Sharing the load: Mex67-Mtr2 cofunctions with Los1 in primary tRNA nuclear export.

Eukaryotic transfer RNAs (tRNAs) are exported from the nucleus, their site of synthesis, to the cytoplasm, their site of function for protein synthesis. The evolutionarily conserved ß-importin family member Los1 (Exportin-t) has been the only exporter known to execute nuclear export of newly transcribed intron-containing pre-tRNAs. Interestingly, LOS1 is unessential in all tested organisms. As tRNA nuclear export is essential, we previously interrogated the budding yeast proteome to identify candidates that function in tRNA nuclear export. Here, we provide molecular, genetic, cytological, and biochemical evidence that the Mex67-Mtr2 (TAP-p15) heterodimer, best characterized for its essential role in mRNA nuclear export, cofunctions with Los1 in tRNA nuclear export. Inactivation of Mex67 or Mtr2 leads to rapid accumulation of end-matured unspliced tRNAs in the nucleus. Remarkably, merely fivefold overexpression of Mex67-Mtr2 can substitute for Los1 in los1Δ cells. Moreover, in vivo coimmunoprecipitation assays with tagged Mex67 document that the Mex67 binds tRNAs. Our data also show that tRNA exporters surprisingly exhibit differential tRNA substrate preferences. The existence of multiple tRNA exporters, each with different tRNA preferences, may indicate that the proteome can be regulated by tRNA nuclear export. Thus, our data show that Mex67-Mtr2 functions in primary nuclear export for a subset of yeast tRNAs.

Control of Saccharomyces cerevisiae pre-tRNA processing by environmental conditions.

tRNA is essential for translation and decoding of the proteome. The yeast proteome responds to stress and tRNA biosynthesis contributes in this response by repression of tRNA transcription and alterations of tRNA modification. Here we report that the stress response also involves processing of pre-tRNA 3' termini. By a combination of Northern analyses and RNA sequencing, we show that upon shift to elevated temperatures and/or to glycerol-containing medium, aberrant pre-tRNAs accumulate in yeast cells. For pre-tRNAUAU(Ile) and pre-tRNAUUU Lys) these aberrant forms are unprocessed at the 5' ends, but they possess extended 3' termini. Sequencing analyses showed that partial 3' processing precedes 5' processing for pre-tRNAUAU(Ile). An aberrant pre-tRNA(Tyr) that accumulates also possesses extended 3' termini, but it is processed at the 5' terminus. Similar forms of these aberrant pre-tRNAs are detected in the rex1Δ strain that is defective in 3' exonucleolytic trimming of pre-tRNAs but are absent in the lhp1Δ mutant lacking 3' end protection. We further show direct correlation between the inhibition of 3' end processing rate and the stringency of growth conditions. Moreover, under stress conditions Rex1 nuclease seems to be limiting for 3' end processing, by decreased availability linked to increased protection by Lhp1. Thus, our data document complex 3' processing that is inhibited by stress in a tRNA-type and condition-specific manner. This stress-responsive tRNA 3' end maturation process presumably contributes to fine-tune the levels of functional tRNA in budding yeast in response to environmental conditions.

Genome-wide screen uncovers novel pathways for tRNA processing and nuclear-cytoplasmic dynamics.

Transfer ribonucleic acids (tRNAs) are essential for protein synthesis. However, key gene products involved in tRNA biogenesis and subcellular movement remain to be discovered. We conducted the first comprehensive unbiased analysis of the role of nearly an entire proteome in tRNA biology and describe 162 novel and 12 previously known Saccharomyces cerevisiae gene products that function in tRNA processing, turnover, and subcellular movement. tRNA nuclear export is of particular interest because it is essential, but the known tRNA exporters (Los1 [exportin-t] and Msn5 [exportin-5]) are unessential. We report that mutations of CRM1 (Exportin-1), MEX67/MTR2 (TAP/p15), and five nucleoporins cause accumulation of unspliced tRNA, a hallmark of defective tRNA nuclear export. CRM1 mutation genetically interacts with los1Δ and causes altered tRNA nuclear-cytoplasmic distribution. The data implicate roles for the protein and mRNA nuclear export machineries in tRNA nuclear export. Mutations of genes encoding actin cytoskeleton components and mitochondrial outer membrane proteins also cause accumulation of unspliced tRNA, likely due to defective splicing on mitochondria. Additional gene products, such as chromatin modification enzymes, have unanticipated effects on pre-tRNA end processing. Thus, this genome-wide screen uncovered putative novel pathways for tRNA nuclear export and extensive links between tRNA biology and other aspects of cell physiology.

Healing for destruction: tRNA intron degradation in yeast is a two-step cytoplasmic process catalyzed by tRNA ligase Rlg1 and 5'-to-3' exonuclease Xrn1.

In eukaryotes and archaea, tRNA splicing generates free intron molecules. Although ∼ 600,000 introns are produced per generation in yeast, they are barely detectable in cells, indicating efficient turnover of introns. Through a genome-wide search for genes involved in tRNA biology in yeast, we uncovered the mechanism for intron turnover. This process requires healing of the 5' termini of linear introns by the tRNA ligase Rlg1 and destruction by the cytoplasmic tRNA quality control 5'-to-3' exonuclease Xrn1, which has specificity for RNAs with 5' monophosphate.