Exploring Splicing Factors In Introns Of Plant Mitochondrial Genes By Using Artificial PPR Protein MAP11

The splicing of the mitochondrial gene intron is different from the nuclear gene.The plant mitochondrial intron splicing mechanism currently has two hypothesis,one is a composite similar to the nuclear gene "nuclear spliceosome",i.e.,forming a mitochondrial intron "mito-spliceosome".However,the components,functions and mechanisms of the complex are unclear.The other is there is no so-called mitochondrial intron "mito-spliceosome",and each splice factor is only distributed over the inner syndrome structure,helping the intron folded to form the correct conformation,and complete the splicing of the intron.These two hypothesis are currently lacking in sufficient experimental evidence.Plant mitochondrial group Ⅱ introns lost self-splicing ability in evolution,requiring a large.number of nucleus-encoded splicing factors to help splicing.Recent studies have found that several RNA binding proteins mainly PentaTricopeptide Repeat(PPR)protein,are involved in mitochondrial intron splicing.PPR is arranged in series by 2 to 30 low-level 35 peptide repeat units.PPR is specifically positioned in mitochondria and chloroplast,mediates a number of aspects of plant organellar RNA metabolism:such as RNA editing,RNA intron splicing,RNA stability and RNA maturation.Each PPR repeating unit forms a right hand super-spiral structure,and a particular amino acid of the repeating unit identifies the mechanism of one RNA base by fifth and 35th amino acids and binds RNA,which specifically depends largely on the amino acids in both positions.The artificial PPR protein constructed by the PPR recognition mechanism exhibits predictable sequences in vitro,which can be used to design artificial proteins and bind specific RNA sequences in plants.In this topic,we designed an artificial PPR protein MAP11 containing 11 PPR repeat sequences based on the PPR repeat motif of the Arabidopsis chloroplast PPR10.The MAP 11 can specifically bind to the non-conserved region sequence of the first intron of the mitochondrial complex I gene nad2.The MAP11 contains a mitochondrial ATPase signal peptide that can be specifically imported into mitochondria.We generated the transgenic plants of MAP11 in Arabidopsis.DNA and mRNA and immunoblot analysis showed specific expression of MAP11 in mitochondria.We subsequently performed the coimmunoprecipitation experiments in Arabidopsis and tobacco using the FLAG antibody.The mass spectrometry showed that MAP 11 specifically binds to nad2 intron 1 sequences,and the associated RNA binding proteins i.e.RNA helicase,pre-RNA splicing factors and PPR proteins are separated.We have conducted a preliminary function analysis.In summary,we conducted an artificial PPR protein to a specific RNA sequence and extracted some related splicing factors,which facilitate the manipulation and analysis of RNA-mediated transcriptional processing functions,which would reveal the intron splicing mechanism of mitochondria.