Structural And Functional Insights Into Erh1p-Mmi1p Complex In Fission Yeast

Gene expression in organisms regulates normal physiological processes.Prokaryotes and eukaryotes utilize different gene expression systems to regulate various physiological activities in vivo.In comparison with prokaryotes,eukaryotes have more complicated gene regulation systems,with cell cycles including meiosis and mitosis for gametes and somatic cell devision,respectively.Transcription,translation and silencing of different genes are involved in cell division.The orderly progress of these processes are the basis for ensuring normal physiological activities in cells.However,the molecular mechanism of these processes still remain unclear.Schizosaccharomyces pombe Erhl,highly conserved homolog of human erh,could interact with Mmil to form Erhl-Mmil complex(EMC).Mmi1,RNA binding protein that only exists in yeast,plays an important role in the elimination of meiotic specific genes.Mmi1 contains low complexity region and a YTH domian at its N-and C-terminus,respectively.In Schizosaccharomyces pombe,Mmi1 YTH domain recognizes determinant of selective removal sequence(DSR,UNAAAC motif repeat)containing meiotic mRNA and subsequently recruit the corresponding complex to eliminate these transcripts avoiding the untimely expression.During entry into meiosis,Mei2-meiRNA complex suppress the activity of Mmi1 to ensure the normal expression of meiotic genes.EMC complex plays an important role in the regulation of related genes and in the formation of heterochromatin.In addition,Erhl-Mmil complex also participates in the interaction with some other complexes and regulate related physiological processes.However,the structure and function of Erhl-Mmil complex still remain enigmatic.Here,amino acid sequence of Mmil from various species were aligned and the conserved sequence were investigated for further interaction studies.Subsequently,Erhl-Mmil complex was expressed in E.Coli,followed by crystallization and resolving the structure of the complex at resolution of 2.7 A(PDB code:6AKJ).Structural analysis of EMC complex reflected Erhl homodimer interact with Mmil via conserved molecular interface at the stoichiometric ratio of 2:2.The conserved molecular interface is mainly composed of hydrophobic residues.Mmi1w112 was speculated as a key residue connecting the two helices at the C-terminal of Mmi195-122,and further insert the hydrophobic pocket of Erh1 and Mmi1 interaction.Mutagenesis analysis reflected that W112A abolished the structure of EMC complex,suggesting that MmilW112 is a crucial residue for the formation and stability of EMC.Meanwhile,Grewal's research group constructed series of mutant strains and performed various functional experiments including co-localization in cells,western blotting,qPCR and ChIP-seq.Taken together,our biochemical analysis and functional assays demonstrated that Mmi1W112 is critical residue for the formation of EMC complex in vivo.Furthermore,it also suggested that EMC plays role in gene silencing and heterochromatin assembly.The thesis is comprised of three chapters.First chapter include research background about the EMC.Second portion contain experimental methods while the third chapter is about the results and discussion of the current project.