Functional Analysis of Sub2 in Prespliceosome Assembly

• Main Authors: Tzu-Hao Wu, 吳梓豪
• Other Authors: Soo-Chen Cheng
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/75059283557744336511

碩士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 105 === Pre-mRNA splicing is one of the most important biological processes in eukaryotic cells.Splicing takes place on a large ribonucleoprotein complex called the spliceosome, which is assembled by sequential binding of five small nuclear RNAs and many protein factors.At the veryearly stage of spliceosome assembly,U1 binds to the 5’ splice site to form the Commitment Complex (CC). Two CCs, CC1 and CC2, were identified.CC2 forms upon the binding of Mud2 and BBP (branch binding protein), which recognizes the 3’ splice site and the branch site,respectively. DEAD boxproteinSub2 has been proposed to play a role in removing Mud2 and BBP fromthepre-mRNA, before or after U2 snRNPbinds to the pre-mRNA to form the pre-spliceosome.However, the mechanismunderlying how Sub2mediatesspliceosome assembly is still a mystery. To unveil this mystery, I constructed a yeast strain to deplete Sub2 in vivo. I also purified recombinant wild-type and dominant-negative Sub2-S247L protein with V5 tag at the N-terminus.However, adding back WT-Sub2 orSub2-S247L protein could not rescue or inhibit the splicing activity under different conditions. SUB2-turn off yeast extracts still provide a tool to study early stage of pre-spliceosome assembly.