| PPR proteins are RNA binding proteins that involved in the RNA metabolism of the mitochondria and the chloroplast.The PPR proteins have a sequence-specific recognition mode of single-stranded RNA,and are closely related with RNA transcription,cut,edit,and stability.The PPR proteins are regulators of mitochondrial proteins,while expression disorder of the PPR proteins will directly lead to a variety of diseases,including cancer,neurodegenerative diseases,diabetes,heart disease,and metabolic syndrome.Study of the expression mechanism of the mitochondrial genes,can offer help for basic treatment of mitochondrial diseases.Schizosaccharomyces pombe as a simple eukaryotic model organism,is also a remarkable mitochondria research model,because fission yeast and mammalian cells are highly similarity both in respiratory physiology and mitochondrial genome.According to the relevant literature,it has been found that S.pombe has 10 PPR proteins,most of which are closely related to the stability and translation of mitochondrial RNA.The function of the Ppr10 is unclear.In this study,S.pombe was used as a model organism to investigate the functions of its Ppr10,for promoting the understanding of the disease which caused by the processing defect of mitochondrial RNA.Hayles J and Kim DU have reported in 2013 and 2010 that the pprll(system name SPAPB1E7.11c)gene is a non-essential gene.But this experiment tried many times but still can not get pprll gene deletion haploid mutant strains.The teaeher Yu Yao of the Fudan University,generously sent pprll gene deletion mutant strains to our laboratory,which is constructed by Kim DU.But after PCR test,this study found that Kim DU failed to build pprll gene deletion mutant strains.And this experiment has proved that the pprll is an essential gene by random spore analysis.In order to further verify the pprll is an essential gene,the plasmid pREP82X-pprll was transformed into diploid strains which contain a chromosomal deletion of pprll.After spore germination we can get viable haploid yeast strains carrying pREP82X-ppr11 plasmid.Thereby further validates the pprll is an essential gene.This experiment eventually proved that the pprll is an essential gene in S.pombe via random spore analysis and plasmid transform.We named the encoded protein of pprll as Ppr11 protein.The reason we named it as Pprll protein is that we found the Pprll protein has a PPR motif by bioinformatics methods.Currently found only 10 PPR proteins In S.pormbe,while the Ppr11 is the 11th PPR protein that has never been reported.We get the Apprll mutation strain based on the original strain NB34-21A,which carrying the ptp1-1 nuclear mutation.The NB34-21A strains allow growth of the S.pombe cells lacking mtDNA.Although we do not yet understand the nature of the nuclear mutations described here that allow growth of S.pombe cells lacking mtDNA,the availability of such strains may prove useful for the analysis of mitochondrial functions in that species as well.In the ?pprll and ?ppr10 mutation strains,an inability to grow on non-fermentation medium is indicative of a strong defect in the mitochondrial respiratory chain.The growth performance of the double mutant was partially restored on EMM medium,indicating their functions are mitochondria-related,and there is a genetic interaction between ppr10 and ppr11.Our laboratory has tagged Ppr10 protein with TAP tag,and the purified protein complex is identified by mass spectrometry,and we found the interaction between Ppr10 and Pprll.Through the Co-immunoprecipitation experiment,our laboratory has proved that there is an interaction between Ppr10 and Ppr11 in vivo.In order to further verify if there is a direct interaction between PprlO and Pprll,we construct the prokaryotic expression of Ppr10 and Pprll fusion protein.Preliminary results showed no detected direct interaction between Pprll and Ppr10 through the Co-Immunoprecipitation experiment. |
PDF Full Text Request