Partial Mechanisms Of Telomere Regulatory Protein Rif1/Rif2 And Nuclear Pore Protein Nup84/Nup170 Mediated Lifespan Regulation Of Saccharomyces Cerevisiae

In the past few decades,the research on aging has achieved unprecedented development,especially through the advancement of biochemical processes and genetic pathways,controlling the aging and aging-related diseases.It is generally believed that aging is the result of a combination of stem cell decline,DNA degradation,dietary psychology,and aging gene activity.Today,there is still no unified theory of aging.Studying aging and aging-related diseases,the ultimate goal is to find the target of drug action to improve human health.Saccharomyces cerevisiae as a single-cell eukaryote,is often used as a model organism for genetic engineering and cell cycle research,because of its ease of cultivation,ease of manipulation,and rapid propagation.Rif1 and Rif2 are two negative regulators of telomere length.Some evidence suggests that Rif1 and Rif2 inhibit the entry of telomerase into telomeres,resulting in shorter telomere length.At present,many scholars believe that telomere length has become a marker of cellular aging.In this study,we found by staining and cloning counting that simultaneous deletion of Rif1 and Rif2 resulted in a significant increase in the lifespan of yeast,and the resistance of the mutant to external stress was also enhanced.However,in earlier experiments,it was found that Rif1 and Rif2 do not regulate yeast lifespan by altering telomere length.Therefore,we analyzed the physicochemical properties,cell localization,structural analysis,and interaction protein prediction of Rif1 and Rif2 through bioinformatics techniques,and provided ideas for further research on the regulatory mechanisms of Rif1 and Rif2.Nuclear pore proteins are a general term of proteins that make up nuclear pore complexes that regulate proteins and nucleic acids in and out of the nucleus.In an early experiment,we performed a rapamycin-sensitive growth assay and found that Nup84 or Nup170 mutants are sensitive to low concentrations of rapamycin treatment,suggesting that Nup84 and Nup170 are directly involved in rapamycin-mediated regulation of S.cerevisiae lifespan.In this study,we performed a series of tests on the longevity and longevity related phenotypes of the two mutants.The results showedthat the two mutants had significantly shorter lifespan and reduced resistance to stress in the external environment after treatment with rapamycin compared to wild-type yeast.In an early experiment,we found that the expression of Atg8 in Nup84 mutant and Nup170 mutant is altered,while Atg8 is a regulator of autophagy.We hypothesized that Nup84 and Nup170 are involved in rapamycin-mediated autophagy in yeast cells.By detecting the alkaline phosphatase activity by the ALP method,we found that the deletion of Atg8,Nup84 or Nup170 resulted in a decrease in intracellular alkaline phosphatase activity,indicating that Nup84 and Nup170 are involved in rapamycin-mediated autophagy.Rim15 is regulating Atg8 transcription.We guess that Nup84 and Nup170 interact with Rim15 to participate in the process of rapamycin regulating yeast autophagy by IP and Western Blot.This study also provides new ideas for studying mechanisms of rapamycin-mediated lifespan regulation.