跳到主要內容

臺灣博碩士論文加值系統

(3.236.84.188) 您好!臺灣時間:2021/08/03 09:11
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:江庭蔚
研究生(外文):Ting-Wei Chiang
論文名稱:酵母菌剪接因子Yju2與Cwc25之功能分析
論文名稱(外文):Functional characterization of Yju2 and Cwc25 in the first catalytic reaction of pre-mRNA splicing
指導教授:鄭淑珍鄭淑珍引用關係
指導教授(外文):Soo-Chen Cheng
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:79
中文關鍵詞:核醣核酸剪接反應剪接體酵母菌
外文關鍵詞:RNA splicingspliceosomeYju2Cwc25yeast
相關次數:
  • 被引用被引用:0
  • 點閱點閱:67
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
Yju2與Cwc25已被證實在活體內與活體外皆為前訊息核醣核酸進行剪接反應所必須,並且參與在Prp2之後第一催化反應不需ATP的步驟。我的研究顯示Yju2主要透過其胺基端與Ntc90間之交互作用與剪接體結合,而Cwc25與剪接體的結合需要在Prp2作用以及Yju2結合之後。在演化上保守的Yju2胺基端對於細胞生長是必須的,並且能夠在去除Yju2之剪接萃取液中部份修復第一催化反應之活性,對於第二催化反應則剩下些微活性,這意謂著其在剪接反應過程中扮演重要的角色。研究顯示在gel mobility shift assays中Yju2能夠與U2小核核醣核酸以及前訊息核醣核酸結合,暗示著其在催化反應期間可能調控U2小核核醣核酸與前訊息核醣核酸結構上之改變。此外,我也發現錳離子能夠增加第一催化反應的效率,尤其當Yju2胺基端存在時更加明顯,這意謂著當剪接體呈現更加動態化時,其能形成發生催化反應之必要結構之機率亦隨之增加,甚至可克服剪接體的部分缺失。研究證實有一抗熱因子HP-X與Yju2以及Cwc25共同參與同一步驟之剪接反應,並可擇一與Yju2或是Cwc25結合,抑或少量與兩者同時結合。此外,當親和性純化之剪接體作用於低濃度的錳離子反應緩衝液時,Cwc25與 HP-X在第一催化反應中之必要性將不再那麼重要。這些結果隱涵著Cwc25與 HP-X可能於第一催化反應期間扮演著促進拉攏5’剪接位與分歧點間距離之輔助角色。
Yju2 and Cwc25 have been demonstrated to be essential for pre-mRNA splicing both in vivo and in vitro, and required for the ATP-independent step of the first catalytic reaction after the action of Prp2. In this study, I have shown that Yju2 is recruited to the spliceosome via the interaction of its N-terminus with Ntc90, and the association of Cwc25 with the spliceosome requires the function of Prp2 and prior association of Yju2. The evolutionarily conserved N-terminal half of Yju2 is essential for cellular growth and able to partially restore the first step activity but barely the second step activity, suggesting its important role in the splicing pathway. I provide evidence that Yju2 is able to bind U2 snRNA and pre-mRNA in gel mobility shift assays, implicating its possible function in mediating the conformational change of U2 and pre-mRNA during the catalytic reaction. Additionally, I have found that Mn2+ is able to increase the efficiency of the first catalytic reaction with a more conspicuous difference in the presence of Yju2-N, suggesting that under circumstances where spliceosome dynamics may increase, the chance of attaining a catalytically competent conformation will correspondingly increase to allow catalysis even though the spliceosome is defective. A heat-resistant factor, HP-X, which is either associated with Yju2 or with Cwc25, or with both in a small amount, has also been shown to be required for the same step. Moreover, the requirement of Cwc25 and HP-X for the first catalytic reaction could be partially relieved when the affinity-purified spliceosome was incubated in the presence of low concentrations of Mn2+. These results have implications for the possible roles of Cwc25 and HP-X in facilitating juxtaposition of the 5’ splice site and the branchpoint during the first catalytic reaction.
Acknowledgements I
摘要 II
Abstract III
Contents IV
Introduction 1
1. The chemical reaction of pre-mRNA splicing 1
2. The determinant of the splice sites 1
3. The constituents of the spliceosome 2
4. Stepwise splicing pathway 6
5. Yju2, Cwc25 and HP-X in the ATP-independent step of the first catalytic reaction. 8
Materials and Methods 11
1. Escherichia coli strains 11
2. Yeast (Saccharomyces cerevisiae) strains 11
3. Oligonucleotides 11
4. Media 12
5. Plasmids 13
6. Purification of recombinant proteins 15
7. Antibodies and reagents 15
8. Preparation of yeast splicing extracts 16
9. In vitro transcription of 32P-labeled RNA 18
10. Assay of in vitro splicing reaction 18
11. Immunodepletion, immunoprecipitation and precipitation of the spliceosome by streptavidin agarose. 19
12. Yeast transformation 20
13. Preparation of biotinylated pre-mRNA 20
14. Western blotting 20
15. Spot assays of yeast 21
16. Yeast two hybrid assays 21
17. Preparation of yeast cell lysates by glass beads 22
18. Preparation of heat-resistant extract fractions (HP) 22
19. Complementation of the affinity-purified spliceosome formed in Yju2- or Cwc25-depleted extracts. 22
20. Electrophoresis mobility shift assay (EMSA) 23
21. UV crosslinking of protein with RNA 23
Results 25
Part A. Domain analysis and functional characterization of Yju2 25
1. The N-terminal domain of Yju2 interacts mainly with Ntc90, while the C-terminal domain interacts only with Ntc77. 25
2. The N-terminal half of Yju2 is essential for cellular growth. 26
3. The N-terminus of Yju2 is able to partially restore the first step activity in Yju2-depleted extracts, but barely the second step activity. 26
4. Mn2+ increases the efficiency of the first catalytic reaction. 27
5. Yju2 has RNA-binding ability. 28
Part B. Functional interactions of Yju2, Cwc25 and HP-X in the first catalytic step of the splicing reaction 29
1. Cwc25 is associated with the spliceosome after Yju2. 29
2. Cwc25, a component of heat-resistant factors, is not sufficient to replace the function of HP in facilitating the first catalytic reaction. 32
3. HP-X is sensitive to proteinase K digestion. 33
4. Cwc25 functions in the final step of the first catalytic reaction after Prp2 and Yju2. 33
5. Yju2, Cwc25 and HP-X are required for the ATP-independent step of the first catalytic reaction. 34
6. The requirements of Cwc25 and HP-X could be partially compensated for in the presence of low levels of Mn2+. 35
Discussion 38
Part A. Domain analysis and functional characterization of Yju2 38
Part B. Functional interactions of Yju2, Cwc25 and HP-X in the first catalytic step of the splicing reaction 41
References 46
Figures 52
Achsel, T., Brahms, H., Kastner, B., Bachi, A., Wilm, M., and Luhrmann, R. (1999). A doughnut-shaped heteromer of human Sm-like proteins binds to the 3'-end of U6 snRNA, thereby facilitating U4/U6 duplex formation in vitro. EMBO J 18, 5789-5802.
Ares, M., Jr., and Weiser, B. (1995). Rearrangement of snRNA structure during assembly and function of the spliceosome. Prog Nucleic Acid Res Mol Biol 50, 131-159.
Battle, D.J., Kasim, M., Yong, J., Lotti, F., Lau, C.K., Mouaikel, J., Zhang, Z., Han, K., Wan, L., and Dreyfuss, G. (2006). The SMN complex: an assembly machine for RNPs. Cold Spring Harb Symp Quant Biol 71, 313-320.
Black, D.L., Chabot, B., and Steitz, J.A. (1985). U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing. Cell 42, 737-750.
Branlant, C., Krol, A., Ebel, J.P., Lazar, E., Haendler, B., and Jacob, M. (1982). U2 RNA shares a structural domain with U1, U4, and U5 RNAs. EMBO J 1, 1259-1265.
Brow, D.A. (2002). Allosteric cascade of spliceosome activation. Annu Rev Genet 36, 333-360.
Brys, A., and Schwer, B. (1996). Requirement for SLU7 in yeast pre-mRNA splicing is dictated by the distance between the branchpoint and the 3' splice site. RNA 2, 707-717.
Chan, S.P., and Cheng, S.C. (2005). The Prp19-associated complex is required for specifying interactions of U5 and U6 with pre-mRNA during spliceosome activation. J Biol Chem 280, 31190-31199.
Chan, S.P., Kao, D.I., Tsai, W.Y., and Cheng, S.C. (2003). The Prp19p-associated complex in spliceosome activation. Science 302, 279-282.
Chen, C.H., Tsai, W.Y., Chen, H.R., Wang, C.H., and Cheng, S.C. (2001). Identification and characterization of two novel components of the Prp19p-associated complex, Ntc30p and Ntc20p. J Biol Chem 276, 488-494.
Chen, C.H., Yu, W.C., Tsao, T.Y., Wang, L.Y., Chen, H.R., Lin, J.Y., Tsai, W.Y., and Cheng, S.C. (2002). Functional and physical interactions between components of the Prp19p-associated complex. Nucleic Acids Res 30, 1029-1037.
Chen, H.R., Tsao, T.Y., Chen, C.H., Tsai, W.Y., Her, L.S., Hsu, M.M., and Cheng, S.C. (1999). Snt309p modulates interactions of Prp19p with its associated components to stabilize the Prp19p-associated complex essential for pre-mRNA splicing. Proc Natl Acad Sci U S A 96, 5406-5411.
Cheng, S.C., and Abelson, J. (1987). Spliceosome assembly in yeast. Genes Dev 1, 1014-1027.
Crotti, L.B., Bacikova, D., and Horowitz, D.S. (2007). The Prp18 protein stabilizes the interaction of both exons with the U5 snRNA during the second step of pre-mRNA splicing. Genes Dev 21, 1204-1216.
Frank, D., and Guthrie, C. (1992). An essential splicing factor, SLU7, mediates 3' splice site choice in yeast. Genes Dev 6, 2112-2124.
Frank, D., Patterson, B., and Guthrie, C. (1992). Synthetic lethal mutations suggest interactions between U5 small nuclear RNA and four proteins required for the second step of splicing. Mol Cell Biol 12, 5197-5205.
Gavin, A.C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, A., Schultz, J., Rick, J.M., Michon, A.M., Cruciat, C.M., et al. (2002). Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141-147.
Ghetti, A., Company, M., and Abelson, J. (1995). Specificity of Prp24 binding to RNA: a role for Prp24 in the dynamic interaction of U4 and U6 snRNAs. RNA 1, 132-145.
Hartmuth, K., Urlaub, H., Vornlocher, H.P., Will, C.L., Gentzel, M., Wilm, M., and Luhrmann, R. (2002). Protein composition of human prespliceosomes isolated by a tobramycin affinity-selection method. Proc Natl Acad Sci U S A 99, 16719-16724.
Hilliker, A.K., Mefford, M.A., and Staley, J.P. (2007). U2 toggles iteratively between the stem IIa and stem IIc conformations to promote pre-mRNA splicing. Genes Dev 21, 821-834.
Horowitz, D.S., and Abelson, J. (1993). A U5 small nuclear ribonucleoprotein particle protein involved only in the second step of pre-mRNA splicing in Saccharomyces cerevisiae. Mol Cell Biol 13, 2959-2970.
James, S.A., Turner, W., and Schwer, B. (2002). How Slu7 and Prp18 cooperate in the second step of yeast pre-mRNA splicing. RNA 8, 1068-1077.
Jones, M.H., Frank, D.N., and Guthrie, C. (1995). Characterization and functional ordering of Slu7p and Prp17p during the second step of pre-mRNA splicing in yeast. Proc Natl Acad Sci U S A 92, 9687-9691.
Jurica, M.S., Licklider, L.J., Gygi, S.R., Grigorieff, N., and Moore, M.J. (2002). Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis. RNA 8, 426-439.
Kambach, C., Walke, S., Young, R., Avis, J.M., de la Fortelle, E., Raker, V.A., Luhrmann, R., Li, J., and Nagai, K. (1999). Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs. Cell 96, 375-387.
Kim, S.H., and Lin, R.J. (1993). Pre-mRNA splicing within an assembled yeast spliceosome requires an RNA-dependent ATPase and ATP hydrolysis. Proc Natl Acad Sci U S A 90, 888-892.
Kim, S.H., and Lin, R.J. (1996). Spliceosome activation by PRP2 ATPase prior to the first transesterification reaction of pre-mRNA splicing. Mol Cell Biol 16, 6810-6819.
Kolb, S.J., Battle, D.J., and Dreyfuss, G. (2007). Molecular functions of the SMN complex. J Child Neurol 22, 990-994.
Konarska, M.M., Grabowski, P.J., Padgett, R.A., and Sharp, P.A. (1985). Characterization of the branch site in lariat RNAs produced by splicing of mRNA precursors. Nature 313, 552-557.
Konarska, M.M., and Sharp, P.A. (1987). Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell 49, 763-774.
Laggerbauer, B., Lauber, J., and Luhrmann, R. (1996). Identification of an RNA-dependent ATPase activity in mammalian U5 snRNPs. Nucleic Acids Res 24, 868-875.
Last, R.L., Maddock, J.R., and Woolford, J.L., Jr. (1987). Evidence for related functions of the RNA genes of Saccharomyces cerevisiae. Genetics 117, 619-631.
Lin, R.J., Lustig, A.J., and Abelson, J. (1987). Splicing of yeast nuclear pre-mRNA in vitro requires a functional 40S spliceosome and several extrinsic factors. Genes Dev 1, 7-18.
Liu, Q., Fischer, U., Wang, F., and Dreyfuss, G. (1997). The spinal muscular atrophy disease gene product, SMN, and its associated protein SIP1 are in a complex with spliceosomal snRNP proteins. Cell 90, 1013-1021.
Liu, Y.C., Chen, H.C., Wu, N.Y., and Cheng, S.C. (2007). A novel splicing factor, Yju2, is associated with NTC and acts after Prp2 in promoting the first catalytic reaction of pre-mRNA splicing. Mol Cell Biol 27, 5403-5413.
Madhani, H.D., and Guthrie, C. (1994). Dynamic RNA-RNA interactions in the spliceosome. Annu Rev Genet 28, 1-26.
Makarov, E.M., Makarova, O.V., Urlaub, H., Gentzel, M., Will, C.L., Wilm, M., and Luhrmann, R. (2002). Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome. Science 298, 2205-2208.
Matera, A.G., and Shpargel, K.B. (2006). Pumping RNA: nuclear bodybuilding along the RNP pipeline. Curr Opin Cell Biol 18, 317-324.
Mayes, A.E., Verdone, L., Legrain, P., and Beggs, J.D. (1999). Characterization of Sm-like proteins in yeast and their association with U6 snRNA. EMBO J 18, 4321-4331.
Moore, M.J., and Sharp, P.A. (1993). Evidence for two active sites in the spliceosome provided by stereochemistry of pre-mRNA splicing. Nature 365, 364-368.
Ohi, M.D., Link, A.J., Ren, L., Jennings, J.L., McDonald, W.H., and Gould, K.L. (2002). Proteomics analysis reveals stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs. Mol Cell Biol 22, 2011-2024.
Patel, S.B., and Bellini, M. (2008). The assembly of a spliceosomal small nuclear ribonucleoprotein particle. Nucleic Acids Res 36, 6482-6493.
Perriman, R.J., and Ares, M., Jr. (2007). Rearrangement of competing U2 RNA helices within the spliceosome promotes multiple steps in splicing. Genes Dev 21, 811-820.
Pikielny, C.W., and Rosbash, M. (1986). Specific small nuclear RNAs are associated with yeast spliceosomes. Cell 45, 869-877.
Ritchie, D.B., Schellenberg, M.J., Gesner, E.M., Raithatha, S.A., Stuart, D.T., and Macmillan, A.M. (2008). Structural elucidation of a PRP8 core domain from the heart of the spliceosome. Nat Struct Mol Biol 15, 1199-1205.
Rocak, S., and Linder, P. (2004). DEAD-box proteins: the driving forces behind RNA metabolism. Nat Rev Mol Cell Biol 5, 232-241.
Rosbash, M., and Seraphin, B. (1991). Who's on first? The U1 snRNP-5' splice site interaction and splicing. Trends Biochem Sci 16, 187-190.
Roy, J., Kim, K., Maddock, J.R., Anthony, J.G., and Woolford, J.L., Jr. (1995). The final stages of spliceosome maturation require Spp2p that can interact with the DEAH box protein Prp2p and promote step 1 of splicing. RNA 1, 375-390.
Ruby, S.W., and Abelson, J. (1988). An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly. Science 242, 1028-1035.
Ruby, S.W., and Abelson, J. (1991). Pre-mRNA splicing in yeast. Trends Genet 7, 79-85.
Ruskin, B., Krainer, A.R., Maniatis, T., and Green, M.R. (1984). Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell 38, 317-331.
Ryan, D.E., and Abelson, J. (2002). The conserved central domain of yeast U6 snRNA: importance of U2-U6 helix Ia in spliceosome assembly. RNA 8, 997-1010.
Salgado-Garrido, J., Bragado-Nilsson, E., Kandels-Lewis, S., and Seraphin, B. (1999). Sm and Sm-like proteins assemble in two related complexes of deep evolutionary origin. EMBO J 18, 3451-3462.
Schwer, B. (2008). A conformational rearrangement in the spliceosome sets the stage for Prp22-dependent mRNA release. Mol Cell 30, 743-754.
Schwer, B., and Gross, C.H. (1998). Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing. EMBO J 17, 2086-2094.
Schwer, B., and Guthrie, C. (1991). PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome. Nature 349, 494-499.
Schwer, B., and Guthrie, C. (1992). A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis. EMBO J 11, 5033-5039.
Shuster, E.O., and Guthrie, C. (1988). Two conserved domains of yeast U2 snRNA are separated by 945 nonessential nucleotides. Cell 55, 41-48.
Silverman, E.J., Maeda, A., Wei, J., Smith, P., Beggs, J.D., and Lin, R.J. (2004). Interaction between a G-patch protein and a spliceosomal DEXD/H-box ATPase that is critical for splicing. Mol Cell Biol 24, 10101-10110.
Singh, R., and Reddy, R. (1989). Gamma-monomethyl phosphate: a cap structure in spliceosomal U6 small nuclear RNA. Proc Natl Acad Sci U S A 86, 8280-8283.
Staley, J.P., and Guthrie, C. (1998). Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell 92, 315-326.
Stevens, S.W., Ryan, D.E., Ge, H.Y., Moore, R.E., Young, M.K., Lee, T.D., and Abelson, J. (2002). Composition and functional characterization of the yeast spliceosomal penta-snRNP. Mol Cell 9, 31-44.
Tanner, N.K., and Linder, P. (2001). DExD/H box RNA helicases: from generic motors to specific dissociation functions. Mol Cell 8, 251-262.
Tarn, W.Y., and Steitz, J.A. (1996). A novel spliceosome containing U11, U12, and U5 snRNPs excises a minor class (AT-AC) intron in vitro. Cell 84, 801-811.
Tsai, R.T., Fu, R.H., Yeh, F.L., Tseng, C.K., Lin, Y.C., Huang, Y.H., and Cheng, S.C. (2005). Spliceosome disassembly catalyzed by Prp43 and its associated components Ntr1 and Ntr2. Genes Dev 19, 2991-3003.
Tsai, R.T., Tseng, C.K., Lee, P.J., Chen, H.C., Fu, R.H., Chang, K.J., Yeh, F.L., and Cheng, S.C. (2007). Dynamic interactions of Ntr1-Ntr2 with Prp43 and with U5 govern the recruitment of Prp43 to mediate spliceosome disassembly. Mol Cell Biol 27, 8027-8037.
Tsai, W.Y., Chow, Y.T., Chen, H.R., Huang, K.T., Hong, R.I., Jan, S.P., Kuo, N.Y., Tsao, T.Y., Chen, C.H., and Cheng, S.C. (1999). Cef1p is a component of the Prp19p-associated complex and essential for pre-mRNA splicing. J Biol Chem 274, 9455-9462.
Tseng, C.K., and Cheng, S.C. (2008). Both catalytic steps of nuclear pre-mRNA splicing are reversible. Science 320, 1782-1784.
Vidal, V.P., Verdone, L., Mayes, A.E., and Beggs, J.D. (1999). Characterization of U6 snRNA-protein interactions. RNA 5, 1470-1481.
Vijayraghavan, U., and Abelson, J. (1990). PRP18, a protein required for the second reaction in pre-mRNA splicing. Mol Cell Biol 10, 324-332.
Wahl, M.C., Will, C.L., and Luhrmann, R. (2009). The spliceosome: design principles of a dynamic RNP machine. Cell 136, 701-718.
Wassarman, D.A., and Steitz, J.A. (1992). Interactions of small nuclear RNA's with precursor messenger RNA during in vitro splicing. Science 257, 1918-1925.
Will, C.L., and Luhrmann, R. (1997). Protein functions in pre-mRNA splicing. Curr Opin Cell Biol 9, 320-328.
Zhang, X., and Schwer, B. (1997). Functional and physical interaction between the yeast splicing factors Slu7 and Prp18. Nucleic Acids Res 25, 2146-2152.
Zhou, Z., Licklider, L.J., Gygi, S.P., and Reed, R. (2002). Comprehensive proteomic analysis of the human spliceosome. Nature 419, 182-185.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top