The Study On The Interaction And Function Of The Tudor-SN Protein And SmB Protein

Objective:TSN domain within the Tudor-SN protein mainly functions in the cytoplasmic assembly of the spliceosomal U snRNPs(Uridine-rich small ribonucleo-proteins) and the nuclear splicing process splicing process of pre-mRNA Crystal structure of the human Tudor-SN TSN domain displays a conserved aromatic cage which hooks methyl groups of U snRNPs. As the vital element of the spliceosome, SmB protein has the symmetrical dimethylarginine (sDMA)-modified arginine and glycine-rich (RG-rich) domains. The present study aims at studying the molecular mechanisms for physical interaction between the Tudor-SN and SmB protein and then further probing the role of Tudor-SN protein in the assembly of the snRNPs.Methods:There are four parts in this study. The first part is to investigate the functional interaction between the Tudor-SN and SmB protein using pulldown and co-immunoprecipitation assays; The second part is to study the influence of key amino acids (Tyr721,Tyr738,Tyr741 or Phe715) within the TSN domain upon the Tudor-SN-SmB binding through the mutagenesis analyses; The third part is to ascertain the specific modification status of SmB protein in the Tudor-SN-SmB complex through the use of the sDMA-specific antibody and protein arginine methyltransferase inhibitor MTA; The fourth part is to observe the change of Tudor-SN-SmB proteins coimmunoprecipitated by the anti-TMG agarose beads in the presence of MTA inhibitor or different expression level of Tudor-SN protein.Result:â‘ Tudor-SN protein interacted with the SmB protein in vivo; and GST-Tudor-SN-TSN/TD binded with the endogenous SmB protein in vitro;â‘¡The weaker binding between the GST-Tudor-SN-TSN-mutant (F715A,Y721A,Y741A,N743A,Y738A,F715A/Y721A,Y318A/Y741A) and the endogenous SmB protein in HeLa cells was detected, compared with GST-Tudor-SN-TSN wild-type (WT) proteins;â‘¢The symmetrical dimethylarginine (sDMA) of Sm protein within the Tudor-SN-SmB complex was verified through the use of the anti-sDMA antibody and the MTA inhibitor;â‘£The Tudor-SN and sDMA-containing endogenous SmB protein were obtained through the anti-TMG-cap agarose beads. And MTA treatment induced the less amount of the SmB protein binding to snRNAs,rather than Tudor-SN snRNAs. In addition, the expression level of the Tudor-SN protein showed positive correlation with the amount of SmB protein co-immunoprecipitated with anti-TMG-cap antibody.Conclusion:Tudor-SN protein is capable of interacting with the SmB protein directly via its TSN or TD domain;â‘¡The conserved amino acids (Phe715,Tyr721,Tyr738 and Tyr741) involved in the TSN cage play an essential role in the Tudor-SN-SmB binding;â‘¢SmB protein in the Tudor-SN-SmB complex shows the symmetrical di-methylarginines modification status;â‘£Tudor-SN protein takes part in and accelerates the cytoplasmic assembly of the spliceosomal U snRNPs.