Functional Analysis of Cwc22 in Saccharomyces cerevisiae

• Main Authors: Tzu-Chi Yeh, 葉子綺
• Other Authors: Soo-Chen Cheng
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/35666348630246153366

碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 96 === In eukaryotic cells, gene expression proceeds through multiple steps, including transcription, RNA maturation and translation. Introns of pre-mRNAs (precursor mRNA) must be removed to become mature mRNAs, which are exported to the cytoplasm. This step is called “pre-mRNA splicing.” The spliceosome is the machinery to perform pre-mRNA splicing. It is composed of the snRNAs (small nuclear RNA) U1, U2, U4/U6, U5 and numerous protein factors. The Prp19-associated complex (NTC，nine-teen complex) is recruited to the spliceosome during spliceosome activative. NTC is required to stabilize the interaction of U5 and U6 with pre-mRNA after U4 is disassociated. NTC contains at least eight proteins: Prp19, Ntc30/ Isy1, Ntc25/ Snt309, Ntc20, Ntc90/ Syf1, Ntc77/ Syf3/ Clf1, Ntc31/ Syf2 and Ntc85/Cef1. Ntc85 has also been identified as Cef1, which has been found to be associated with a complex known as CWC (complexed with Cef1). In this thesis, I studied a component of the CWC complex Cwc22. Cwc22 was required for pre-mRNA splicing both in vivo and in vitro. Cwc22 was not tightly associated with NTC and interacted with Cwc22, Ntc30, Prp45, Ntr1 and Cwc25, suggesting that Cwc22 is not a component of NTC. Yeast extracts depleted of Cwc22 could be complemented by recombinant Cwc22, suggesting that Cwc22 is not tightly associated with other splicing factors. During spliceosome assembly, Cwc22 was associated with the spliceosome after binding of NTC and remained associated until the reaction was complete. Further analysis revealed that Cwc22 was involved in the ATP-dependent step of the first transesterification. My study demonstrates that Cwc22 acted in an ATP-dependent, but Prp2-independent manner in the first catalytic step.