Functional Role of Prp5 in Prespliceosome Formation

• Main Authors: Wen-Wei Liang, 梁雯薇
• Other Authors: Soo-Chen Cheng
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/66777680640670438718

碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 99 === The DExD/H-box Prp5 protein has been demonstrated to be required for the prespliceosome formation via its ATP-dependent and ATP-independent activities. Functional roles of Prp5 in later steps of the spliceosome pathway and in splicing fidelity control have also been implicated. In this thesis, I examined whether the functional role of Prp5 in splicing is restricted to prespliceosome formation. For NTC-depleted or Spp2-depleted extracts, Prp5 depletion did not affect complementation of purified NTC or recombinant Spp2, demonstrating that Prp5 is not required for steps after NTC-mediated spliceosome activation. In contrast to previous reports, I found that formation of the prespliceosome in Cus2-depleted extracts requires the ATPase activity of Prp5, suggesting an additional ATP-dependent activity of Prp5 besides Cus2 removal. The association of Prp5 with the spliceosome is transitory that could not be detected by immunoprecipitation or pull-down assays. However, I found that Prp5 could stably associate with the spliceosome formed on pre-mRNA with branch site mutation (BS-spliceosome). The stabilization of Prp5 with the BS-spliceosome was ATP-independent, and the extent of stabilization was inversely proportional to the splicing efficiency. By UV-crosslinking, U2 snRNA was shown to directly interact with Prp5. The association of Prp5 with the BS-spliceosome was dependent on ATP, the ATPase activity of Prp5, and U2 snRNP. Moreover, the recruitment of tri-snRNPs was impeded, which may result from stalling of Prp5.