RESUMO
Fcp1 is a protein phosphatase that facilitates transcription elongation and termination by dephosphorylating the C-terminal domain of RNA polymerase II. High-throughput genetic screening and gene expression profiling of fcp1 mutants revealed a novel connection to Cdk8, the Mediator complex kinase subunit, and Skn7, a key transcription factor in the oxidative stress response pathway. Briefly, Skn7 was enriched as a regulator of genes whose mRNA levels were altered in fcp1 and cdk8Δ mutants and was required for the suppression of fcp1 mutant growth defects by loss of CDK8 under oxidative stress conditions. Targeted analysis revealed that mutating FCP1 decreased Skn7 mRNA and protein levels as well as its association with target gene promoters but paradoxically increased the mRNA levels of Skn7-dependent oxidative stress-induced genes (TRX2 and TSA1) under basal and induced conditions. The latter was in part recapitulated via chemical inhibition of transcription in WT cells, suggesting that a combination of transcriptional and posttranscriptional effects underscored the increased mRNA levels of TRX2 and TSA1 observed in the fcp1 mutant. Interestingly, loss of CDK8 robustly normalized the mRNA levels of Skn7-dependent genes in the fcp1 mutant background and also increased Skn7 protein levels by preventing its turnover. As such, our work suggested that loss of CDK8 could overcome transcriptional and/or posttranscriptional alterations in the fcp1 mutant through its regulatory effect on Skn7. Furthermore, our work also implicated FCP1 and CDK8 in the broader response to environmental stressors in yeast.
Assuntos
Quinase 8 Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Estresse Oxidativo , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Quinase 8 Dependente de Ciclina/genética , Proteínas de Ligação a DNA/genética , Peroxidases/genética , Peroxidases/metabolismo , Fosfoproteínas Fosfatases/genética , Estabilidade Proteica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
BACKGROUND: To reduce person-to-person contact, the COVID-19 pandemic has driven a massive shift to virtual care. Defined as the use of technology (synchronous or asynchronous) to support communication between health care providers and patients, rural-urban differences in virtual care are relatively unexplored. OBJECTIVE: The 2-fold purpose of this study was to examine rural and urban virtual care access, use, and satisfaction during the pandemic and to identify any unmet needs. METHODS: This study was a cross-sectional online survey exploring virtual care among rural and urban adults in summer 2021 using a combination of fixed and open-ended response options. Quantitative data were analyzed using both descriptive and inferential statistics, and qualitative data were analyzed using inductive thematic content analysis. RESULTS: Overall, 501 (373, 74.4% female; age range 19-86 years; 237, 47.3% rural-living) Western Canadians completed the survey. Virtual care use was high among both rural (171/237, 72.2%) and urban (188/264, 71.2%) participants, with over one-half (279/501, 55.7%) reporting having only started to use virtual care since the pandemic. The self-reported need for mental health programs and services increased during the pandemic, compared with prior for both rural and urban participants. Among virtual care users, interest in its continuation was high. Our analysis also shows that internet quality (all P<.05) and eHealth literacy (all P<.001) were positively associated with participants' perceptions of virtual care usefulness, ease of use, and satisfaction, with no rural-urban differences. Rural participants were less likely to have used video in communicating with doctors or health care providers, compared with urban participants (P<.001). When describing unmet needs, participants described a (1) lack of access to care, (2) limited health promotion and prevention options, and (3) lack of mental health service options. CONCLUSIONS: The increased demand for and use of virtual care may reflect increased availability and a lack of alternatives due to limited in-person services during the COVID-19 pandemic, so a balance between virtual care and in-person care is important to consider postpandemic. Further, ensuring availability of high-speed internet and education to support patients will be important for providing accessible and effective virtual care, especially for rural residents.
RESUMO
Cdk8 of the RNA polymerase II mediator kinase complex regulates gene expression by phosphorylating sequence-specific transcription factors. This function is conserved amongst eukaryotes, but the signals and mechanisms regulating Cdk8 activity and phosphorylation of its substrates are unknown. Full induction of the GAL genes in yeast requires phosphorylation of the transcriptional activator Gal4 by Cdk8. We used a screen to identify regulators of the Cdk8-dependent phosphorylation on Gal4, from which we identified multiple mutants with defects in TORC1 signaling. One mutant, designated gal four throttle 1 (gft1) was identified as a recessive allele of hom3, encoding aspartokinase, and mutations in hom3 caused effects typical of inhibition of TORC1, including rapamycin sensitivity and enhanced nuclear localization of the TORC1-responsive transcription factor Gat1. Mutations in hom3 also inhibit phosphorylation of Gal4 in vivo at the Cdk8-dependent site on Gal4, as did mutations of tor1, but these mutations did not affect activity of Cdk8 assayed in vitro. Disruption of cdc55, encoding a regulatory subunit of the TORC1-regulated protein phosphatase PP2A, suppressed the effect of hom3 and tor1 mutations on GAL expression, and also restored phosphorylation of Gal4 at the Cdk8-dependent site in vivo. These observations demonstrate that TORC1 signaling regulates GAL induction through the activity of PP2A/Cdc55 and suggest that Cdk8-dependent phosphorylation of Gal4 is opposed by PP2A/Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation.