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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭淑珍(Soo-Chen Cheng) | |
| dc.contributor.author | Ching-Yang Kao | en |
| dc.contributor.author | 高景揚 | zh_TW |
| dc.date.accessioned | 2021-06-16T05:51:00Z | - |
| dc.date.available | 2022-07-24 | |
| dc.date.copyright | 2020-07-28 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-07-24 | |
| dc.identifier.citation | Abovich, N., Liao, X.C., and Rosbash, M. (1994). The yeast MUD2 protein: an interaction with PRP11 defines a bridge between commitment complexes and U2 snRNP addition. Genes Dev 8, 843-854. Abovich, N., and Rosbash, M. (1997). Cross-intron bridging interactions in the yeast commitment complex are conserved in mammals. Cell 89, 403-412. Arenas, J.E., and Abelson, J.N. (1997). Prp43: An RNA helicase-like factor involved in spliceosome disassembly. Proc Natl Acad Sci U S A 94, 11798-11802. Aslanzadeh, V., Huang, Y., Sanguinetti, G., and Beggs, J.D. (2018). Transcription rate strongly affects splicing fidelity and cotranscriptionality in budding yeast. Genome Res 28, 203-213. Berg, M.G., Singh, L.N., Younis, I., Liu, Q., Pinto, A.M., Kaida, D., Zhang, Z., Cho, S., Sherrill-Mix, S., Wan, L., et al. (2012). U1 snRNP determines mRNA length and regulates isoform expression. Cell 150, 53-64. Berget, S.M., Moore, C., and Sharp, P.A. (1977). Spliced segments at the 5' terminus of adenovirus 2 late mRNA. Proc Natl Acad Sci U S A 74, 3171-3175. Berglund, J.A., Chua, K., Abovich, N., Reed, R., and Rosbash, M. (1997). The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC. Cell 89, 781-787. Bringmann, P., Appel, B., Rinke, J., Reuter, R., Theissen, H., and Luhrmann, R. (1984). Evidence for the existence of snRNAs U4 and U6 in a single ribonucleoprotein complex and for their association by intermolecular base pairing. EMBO J 3, 1357-1363. Burgess, S.M., and Guthrie, C. (1993). A mechanism to enhance mRNA splicing fidelity: the RNA-dependent ATPase Prp16 governs usage of a discard pathway for aberrant lariat intermediates. Cell 73, 1377-1391. Carrocci, T.J., Zoerner, D.M., Paulson, J.C., and Hoskins, A.A. (2017). SF3b1 mutations associated with myelodysplastic syndromes alter the fidelity of branchsite selection in yeast. Nucleic Acids Res 45, 4837-4852. Castanotto, D., and Rossi, J.J. (1998). Cooperative interaction of branch signals in the actin intron of Saccharomyces cerevisiae. Nucleic Acids Res 26, 4137-4145. Cech, T.R. (1986). The generality of self-splicing RNA: relationship to nuclear mRNA splicing. Cell 44, 207-210. Cellini, A., Parker, R., McMahon, J., Guthrie, C., and Rossi, J. (1986). Activation of a cryptic TACTAAC box in the Saccharomyces cerevisiae actin intron. Mol Cell Biol 6, 1571-1578. Chen, H.C., and Cheng, S.C. (2012). Functional roles of protein splicing factors. Bioscience reports 32, 345-359. Cheng, S.C., and Abelson, J. (1987). Spliceosome assembly in yeast. Genes Dev 1, 1014-1027. Chow, L.C., Gelinas, R.E., Broker, T.R., and Roberts, R.J. (2000). An amazing sequence arrangement at the 5' ends of adenovirus 2 messenger RNA. 1977. Reviews in medical virology 10, 362-371; discussion 355-366. Chung, C.S., Tseng, C.K., Lai, Y.H., Wang, H.F., Newman, A.J., and Cheng, S.C. (2019). Dynamic protein-RNA interactions in mediating splicing catalysis. Nucleic Acids Res 47, 899-910. Company, M., Arenas, J., and Abelson, J. (1991). Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes. Nature 349, 487-493. Cordin, O., and Beggs, J.D. (2013). RNA helicases in splicing. RNA biology 10, 83-95. Cordin, O., Tanner, N.K., Doere, M., Linder, P., and Banroques, J. (2004). The newly discovered Q motif of DEAD-box RNA helicases regulates RNA-binding and helicase activity. EMBO J 23, 2478-2487. Darman, R.B., Seiler, M., Agrawal, A.A., Lim, K.H., Peng, S., Aird, D., Bailey, S.L., Bhavsar, E.B., Chan, B., Colla, S., et al. (2015). Cancer-Associated SF3B1 Hotspot Mutations Induce Cryptic 3' Splice Site Selection through Use of a Different Branch Point. Cell reports 13, 1033-1045. Fabrizio, P., Dannenberg, J., Dube, P., Kastner, B., Stark, H., Urlaub, H., and Luhrmann, R. (2009). The evolutionarily conserved core design of the catalytic activation step of the yeast spliceosome. Mol Cell 36, 593-608. Fackenthal, J.D., Cartegni, L., Krainer, A.R., and Olopade, O.I. (2002). BRCA2 T2722R is a deleterious allele that causes exon skipping. American journal of human genetics 71, 625-631. Fairman-Williams, M.E., Guenther, U.P., and Jankowsky, E. (2010). SF1 and SF2 helicases: family matters. Curr Opin Struct Biol 20, 313-324. Fong, N., Kim, H., Zhou, Y., Ji, X., Qiu, J., Saldi, T., Diener, K., Jones, K., Fu, X.D., and Bentley, D.L. (2014). Pre-mRNA splicing is facilitated by an optimal RNA polymerase II elongation rate. Genes Dev 28, 2663-2676. 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. Gorbalenya, A.E., and Koonin, E.V. (1993). Helicases: amino acid sequence comparisons and structure-function relationships. Current Opinion in Structural Biology 3, 419-429. Gozani, O., Potashkin, J., and Reed, R. (1998). A potential role for U2AF-SAP 155 interactions in recruiting U2 snRNP to the branch site. Mol Cell Biol 18, 4752-4760. Green, M.R. (1986). Pre-mRNA splicing. Annu Rev Genet 20, 671-708. Hashimoto, C., and Steitz, J.A. (1984). U4 and U6 RNAs coexist in a single small nuclear ribonucleoprotein particle. Nucleic Acids Res 12, 3283-3293. 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. Igel, A.H., and Ares, M., Jr. (1988). Internal sequences that distinguish yeast from metazoan U2 snRNA are unnecessary for pre-mRNA splicing. Nature 334, 450-453. Jablonka, S., Holtmann, B., Meister, G., Bandilla, M., Rossoll, W., Fischer, U., and Sendtner, M. (2002). Gene targeting of Gemin2 in mice reveals a correlation between defects in the biogenesis of U snRNPs and motoneuron cell death. Proc Natl Acad Sci U S A 99, 10126-10131. Jacewicz, A., Chico, L., Smith, P., Schwer, B., and Shuman, S. (2015). Structural basis for recognition of intron branchpoint RNA by yeast Msl5 and selective effects of interfacial mutations on splicing of yeast pre-mRNAs. RNA 21, 401-414. Jurica, M.S., and Moore, M.J. (2003). Pre-mRNA splicing: awash in a sea of proteins. Mol Cell 12, 5-14. Kaur, H., Groubert, B., Paulson, J.C., McMillan, S., and Hoskins, A.A. (2020). Impact of cancer-associated mutations in Hsh155/SF3b1 HEAT repeats 9-12 on pre-mRNA splicing in Saccharomyces cerevisiae. PLoS One 15, e0229315. Kistler, A.L., and Guthrie, C. (2001). Deletion of MUD2, the yeast homolog of U2AF65, can bypass the requirement for sub2, an essential spliceosomal ATPase. Genes Dev 15, 42-49. Konarska, M.M., and Sharp, P.A. (1987). Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell 49, 763-774. Kuwasako, K., He, F., Inoue, M., Tanaka, A., Sugano, S., Guntert, P., Muto, Y., and Yokoyama, S. (2006). Solution structures of the SURP domains and the subunit-assembly mechanism within the splicing factor SF3a complex in 17S U2 snRNP. Structure 14, 1677-1689. Lardelli, R.M., Thompson, J.X., Yates, J.R., 3rd, and Stevens, S.W. (2010). Release of SF3 from the intron branchpoint activates the first step of pre-mRNA splicing. RNA 16, 516-528. Lee, Y., and Rio, D.C. (2015). Mechanisms and Regulation of Alternative Pre-mRNA Splicing. Annual review of biochemistry 84, 291-323. Liang, W.W., and Cheng, S.C. (2015). A novel mechanism for Prp5 function in prespliceosome formation and proofreading the branch site sequence. Genes Dev 29, 81-93. Libri, D., Graziani, N., Saguez, C., and Boulay, J. (2001). Multiple roles for the yeast SUB2/yUAP56 gene in splicing. Genes Dev 15, 36-41. Liu, H.L., and Cheng, S.C. (2012). The interaction of Prp2 with a defined region of the intron is required for the first splicing reaction. Mol Cell Biol 32, 5056-5066. Liu, H.X., Cartegni, L., Zhang, M.Q., and Krainer, A.R. (2001). A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes. Nat Genet 27, 55-58. Luco, R.F., Allo, M., Schor, I.E., Kornblihtt, A.R., and Misteli, T. (2011). Epigenetics in alternative pre-mRNA splicing. Cell 144, 16-26. Luhrmann, R., Kastner, B., and Bach, M. (1990). Structure of spliceosomal snRNPs and their role in pre-mRNA splicing. Biochim Biophys Acta 1087, 265-292. Maeder, C., Kutach, A.K., and Guthrie, C. (2009). ATP-dependent unwinding of U4/U6 snRNAs by the Brr2 helicase requires the C terminus of Prp8. Nat Struct Mol Biol 16, 42-48. Maquat, L.E., and Carmichael, G.G. (2001). Quality control of mRNA function. Cell 104, 173-176. Mayerle, M., and Guthrie, C. (2018). Brr2 is a splicing fidelity factor. bioRxiv. McPheeters, D.S., Fabrizio, P., and Abelson, J. (1989). In vitro reconstitution of functional yeast U2 snRNPs. Genes Dev 3, 2124-2136. Minocha, R., Popova, V., Kopytova, D., Misiak, D., Huttelmaier, S., Georgieva, S., and Strasser, K. (2018). Mud2 functions in transcription by recruiting the Prp19 and TREX complexes to transcribed genes. Nucleic Acids Res 46, 9749-9763. Mount, S.M., Pettersson, I., Hinterberger, M., Karmas, A., and Steitz, J.A. (1983). The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro. Cell 33, 509-518. Nilsen, T.W., and Graveley, B.R. (2010). Expansion of the eukaryotic proteome by alternative splicing. Nature 463, 457-463. Nojima, T., Rebelo, K., Gomes, T., Grosso, A.R., Proudfoot, N.J., and Carmo-Fonseca, M. (2018). RNA Polymerase II Phosphorylated on CTD Serine 5 Interacts with the Spliceosome during Co-transcriptional Splicing. Mol Cell 72, 369-379.e364. Oh, J.M., Venters, C.C., Di, C., Pinto, A.M., Wan, L., Younis, I., Cai, Z., Arai, C., So, B.R., Duan, J., et al. (2020). U1 snRNP regulates cancer cell migration and invasion in vitro. Nature communications 11, 1. Padgett, R.A., Konarska, M.M., Grabowski, P.J., Hardy, S.F., and Sharp, P.A. (1984). Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors. Science 225, 898-903. Parker, R., Siliciano, P.G., and Guthrie, C. (1987). Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA. Cell 49, 229-239. Pena, V., Jovin, S.M., Fabrizio, P., Orlowski, J., Bujnicki, J.M., Luhrmann, R., and Wahl, M.C. (2009). Common design principles in the spliceosomal RNA helicase Brr2 and in the Hel308 DNA helicase. Mol Cell 35, 454-466. Perriman, R., and Ares, M., Jr. (2000). ATP can be dispensable for prespliceosome formation in yeast. Genes Dev 14, 97-107. Perriman, R., Barta, I., Voeltz, G.K., Abelson, J., and Ares, M., Jr. (2003). ATP requirement for Prp5p function is determined by Cus2p and the structure of U2 small nuclear RNA. Proc Natl Acad Sci U S A 100, 13857-13862. 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. Query, C.C., and Konarska, M.M. (2004). Suppression of multiple substrate mutations by spliceosomal prp8 alleles suggests functional correlations with ribosomal ambiguity mutants. Mol Cell 14, 343-354. Raghunathan, P.L., and Guthrie, C. (1998a). RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2. Curr Biol 8, 847-855. Raghunathan, P.L., and Guthrie, C. (1998b). A spliceosomal recycling factor that reanneals U4 and U6 small nuclear ribonucleoprotein particles. Science 279, 857-860. Rain, J.C., and Legrain, P. (1997). In vivo commitment to splicing in yeast involves the nucleotide upstream from the branch site conserved sequence and the Mud2 protein. EMBO J 16, 1759-1771. Rain, J.C., Rafi, Z., Rhani, Z., Legrain, P., and Krämer, A. (1998). Conservation of functional domains involved in RNA binding and protein-protein interactions in human and Saccharomyces cerevisiae pre-mRNA splicing factor SF1. RNA 4, 551-565. Raker, V.A., Plessel, G., and Luhrmann, R. (1996). The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro. EMBO J 15, 2256-2269. Rauhut, R., Fabrizio, P., Dybkov, O., Hartmuth, K., Pena, V., Chari, A., Kumar, V., Lee, C.T., Urlaub, H., Kastner, B., et al. (2016). Molecular architecture of the Saccharomyces cerevisiae activated spliceosome. Science 353, 1399-1405. Rondon, A.G., Jimeno, S., Garcia-Rubio, M., and Aguilera, A. (2003). Molecular evidence that the eukaryotic THO/TREX complex is required for efficient transcription elongation. J Biol Chem 278, 39037-39043. 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. Ruskin, B., Zamore, P.D., and Green, M.R. (1988). A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell 52, 207-219. Schneider, C., Agafonov, D.E., Schmitzova, J., Hartmuth, K., Fabrizio, P., and Luhrmann, R. (2015). Dynamic Contacts of U2, RES, Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes. PLoS Genet 11, e1005539. Schwer, B. (2008). A conformational rearrangement in the spliceosome sets the stage for Prp22-dependent mRNA release. Mol Cell 30, 743-754. Seraphin, B., and Rosbash, M. (1989). Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing. Cell 59, 349-358. Sharp, P.A. (1985). On the origin of RNA splicing and introns. Cell 42, 397-400. Shuster, E.O., and Guthrie, C. (1988). Two conserved domains of yeast U2 snRNA are separated by 945 nonessential nucleotides. Cell 55, 41-48. Small, E.C., Leggett, S.R., Winans, A.A., and Staley, J.P. (2006). The EF-G-like GTPase Snu114p regulates spliceosome dynamics mediated by Brr2p, a DExD/H box ATPase. Mol Cell 23, 389-399. So, B.R., Di, C., Cai, Z., Venters, C.C., Guo, J., Oh, J.M., Arai, C., and Dreyfuss, G. (2019). A Complex of U1 snRNP with Cleavage and Polyadenylation Factors Controls Telescripting, Regulating mRNA Transcription in Human Cells. Mol Cell 76, 590-599.e594. Sontheimer, E.J. (1994). Site-specific RNA crosslinking with 4-thiouridine. Mol Biol Rep 20, 35-44. Staley, J.P., and Guthrie, C. (1998). Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell 92, 315-326. Staley, J.P., and Guthrie, C. (1999). An RNA switch at the 5' splice site requires ATP and the DEAD box protein Prp28p. Mol Cell 3, 55-64. Tang, Q., Rodriguez-Santiago, S., Wang, J., Pu, J., Yuste, A., Gupta, V., Moldon, A., Xu, Y.Z., and Query, C.C. (2016). SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing. Genes Dev 30, 2710-2723. Tanner, N.K., Cordin, O., Banroques, J., Doere, M., and Linder, P. (2003). The Q motif: a newly identified motif in DEAD box helicases may regulate ATP binding and hydrolysis. Mol Cell 11, 127-138. Tseng, C.K., Liu, H.L., and Cheng, S.C. (2011). DEAH-box ATPase Prp16 has dual roles in remodeling of the spliceosome in catalytic steps. RNA 17, 145-154. Wahl, M.C., Will, C.L., and Luhrmann, R. (2009). The spliceosome: design principles of a dynamic RNP machine. Cell 136, 701-718. Wang, J., Pegoraro, E., Menegazzo, E., Gennarelli, M., Hoop, R.C., Angelini, C., and Hoffman, E.P. (1995). Myotonic dystrophy: evidence for a possible dominant-negative RNA mutation. Hum Mol Genet 4, 599-606. Warkocki, Z., Odenwalder, P., Schmitzova, J., Platzmann, F., Stark, H., Urlaub, H., Ficner, R., Fabrizio, P., and Luhrmann, R. (2009). Reconstitution of both steps of Saccharomyces cerevisiae splicing with purified spliceosomal components. Nat Struct Mol Biol 16, 1237-1243. Wassarman, D.A. (1993). Psoralen crosslinking of small RNAs in vitro. Mol Biol Rep 17, 143-151. Winston, F., Chumley, F., and Fink, G.R. (1983). Eviction and transplacement of mutant genes in yeast. Methods Enzymol 101, 211-228. Xu, Y.Z., and Query, C.C. (2007). Competition between the ATPase Prp5 and branch region-U2 snRNA pairing modulates the fidelity of spliceosome assembly. Mol Cell 28, 838-849. Yan, C., Wan, R., Bai, R., Huang, G., and Shi, Y. (2016). Structure of a yeast activated spliceosome at 3.5 A resolution. Science 353, 904-911. Zamore, P.D., and Green, M.R. (1989). Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor. Proc Natl Acad Sci U S A 86, 9243-9247. Zhang, L., Xu, T., Maeder, C., Bud, L.O., Shanks, J., Nix, J., Guthrie, C., Pleiss, J.A., and Zhao, R. (2009). Structural evidence for consecutive Hel308-like modules in the spliceosomal ATPase Brr2. Nat Struct Mol Biol 16, 731-739. Zhang, M., and Green, M.R. (2001). Identification and characterization of yUAP/Sub2p, a yeast homolog of the essential human pre-mRNA splicing factor hUAP56. Genes Dev 15, 30-35. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56827 | - |
| dc.description.abstract | 前驅訊息核糖核酸 (pre-mRNA) 剪接反應是由U1結合在5端剪接位，以及Msl5-Mud2異二聚體與分歧點 (branchpoint) 的結合而開始的，從而形成委任複合體 (Commitment complex)。接著由DEAD-box蛋白Prp5協助U2 小核醣核酸複合體與前驅訊息核糖核酸的結合，形成前剪接體。在形成剪接體的過程中，U2 如何被引導至前驅訊息核糖核酸，如何替換Msl5-Mud2，以及Prp5如何脫離的詳細機制尚不清楚。在先前的研究中，我們透過使用分歧點突變的前驅訊息核糖核酸，發現一個含有含有Prp5新的中間複合體，FIC (Prp five-associated intermediate complex )。在此篇研究中，藉由免疫沉澱與化學交叉鏈結分析，我發現Msl5仍然與FIC結合，並且主要的結合位置為分歧點上游的隱蔽結合位 (CBS, Cryptic binding site)。在FIC上，U2 並未與突變的分歧點形成鹼基配對，反而是與分歧點下游區域形成配對。CBS的存在與其相對於分歧點的位置，對於委任複合體的形成非常重要，CBS的缺失也會影響U2與前驅訊息核糖核酸的結合。在剪接反應中使用能夠與突變分歧點完整互補的突變U2小核醣核酸，我發現恢復U2與突變分歧點之間的鹼基配對可促進Prp5從FIC分離，並且挽救剪接缺陷。當我將分歧點下游的序列進行點突變，並增強此區域與U2的配對強度，會降低剪接反應的速率。此結果暗示著在前剪接體形成的過程中，U2可能被加載到分歧點的下游，並沿著3端至5端的方向掃描前驅訊息核糖核酸來搜索分歧點。本研究的結果顯示，Msl5與前驅訊息核糖核酸的正確相互作用對於U2的結合很重要，我也發現Prp5可能是由於U2與分歧點進行鹼基配對後，U2的結構變化而分離，進而推動後續剪接反應的進行。 | zh_TW |
| dc.description.abstract | Splicing of pre-mRNA is initiated by binding of U1 snRNP to the 5’ splice site and of Msl5-Mud2 heterodimer to the branch site (BS), together forming the commitment complex (CC). Subsequent binding of U2 snRNP to the pre-mRNA, facilitated by DEAD-box protein Prp5, forms the prespliceosome. How U2 is recruited to the pre-mRNA to replace Msl5-Mud2 and what triggers dissociation of Prp5 during prespliceosome formation is unknown. The research group of Dr. Soo-Chen Cheng previously identified a prespliceosome precursor complex using BS-mutated ACT1 pre-mRNA that causes arrest of Prp5 on the spliceosome, forming a complex called FIC (for Prp five-associated intermediate complex). By immunoprecipitation and cross-linking analyses, I show that Msl5 stalls on the FIC and binds mainly at the upstream cryptic branch site sequence (CBS). In contrast, instead of interacting with the BS, U2 interacts with sequences downstream of it. Deletion of the CBS weakens binding of Msl5 and U2 to BS-mutated pre-mRNA, and the relative position of the CBS to the mutated BS is important for recruitment of U2 snRNP. Restoring base-pairing between U2 and the mutated BS promotes dissociation of Prp5 from the FIC and rescues the splicing defect in a CBS-dependent manner. Mutating sequences downstream of the mutated BS to enhance the pairing strength between U2 and the pre-mRNA reduced the rate of the splicing reaction, suggesting that U2 may be loaded downstream of the BS and then seeks the BS along the pre-mRNA. My results demonstrate that proper interaction of Msl5 with the pre-mRNA is important for U2 recruitment, and that Prp5 dissociates possibly due to a conformational change of U2 snRNA after base-pairing with the BS to drive the reaction forward. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T05:51:00Z (GMT). No. of bitstreams: 1 U0001-2307202017581900.pdf: 7944887 bytes, checksum: 1cbee94d68ff05e8ecf79fd4909157ad (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 口試委員會審定書 # 誌謝 i 中文摘要 ii ABSTRACT iii CONTENTS iv Chapter 1 Introduction 1 1.1 Overview of pre-mRNA splicing 1 1.2 The pre-mRNA splicing cycle 2 1.3 Small nuclear ribonucleoprotein particles (snRNPs) 3 1.4 DExD/H-box proteins 6 1.5 Mechanism of prespliceosome assembly 7 1.6 Effects of branch site mutations on pre-mRNA splicing 9 Chapter 2 Materials and Methods 11 2.1 Escherichia coli strains 11 2.2 Saccharomyces cerevisiae strains 11 2.3 Oligonucleotides 12 2.4 Plasmids 15 2.5 Culture media and plates 15 2.6 Competent cell preparation 16 2.7 E. coli transformation 17 2.8 Induction of recombinant protein 18 2.9 Purification of recombinant protein 18 2.10 Antibodies 19 2.11 Yeast transformation 19 2.12 Construction of the BJ-U2S strain 19 2.13 Yeast splicing extract 20 2.14 Western blotting 21 2.15 Northern blotting 22 2.16 In vitro transcription 22 2.17 In vitro splicing assay 23 2.18 Immunodepletion 24 2.19 Immunoprecipitation of the spliceosome 24 2.20 4sU cross-linking 25 2.21 Psoralen cross-linking 26 2.22 254 nm UV cross-linking 27 2.23 RNaseH protection assay 28 2.24 Primer extension 28 2.25 Denatured RNA polyacrylamide gel electrophoresis 29 Chapter 3 Results 30 3.1 The FIC assumes a different structure from the prespliceosome 30 3.2 Msl5 is associated with the FIC formed on U257G pre-mRNA 32 3.3 Msl5 interacts with an upstream cryptic branch site on the FIC 33 3.4 U2 does not base pair with the mutated branch site on the FIC 34 3.5 Dynamic interaction of U2 with the pre-mRNA during prespliceosome formation 36 3.6 Restoring U2-BS base pairing promotes the release of Prp5 37 3.7 Position effect of the CBS on FIC formation 37 3.8 U2-A36C-guided usage of the branchpoint on U257G pre-mRNA 39 3.9 U2 might be loaded on the pre-mRNA downstream of the branch site after being recruited to the spliceosome 41 3.10 A model for the mechanism of prespliceosome formation 42 Chapter 4 Discussion 44 4.1 The FIC is a true intermediate before prespliceosome formation 44 4.2 Correct U2-BS base-pairing is required for the release of Prp5 45 4.3 U2 interacts dynamically with the intron during prespliceosome formation 46 4.4 Relative position of CBS to the branch site is important for splicing reaction 47 4.5 Recruitment of U2 to the 3’ region of the branch site 48 4.6 Conclusion 49 Chapter 5 References 50 Chapter 6 Figures 60 Chapter 7 Appendix 102 | |
| dc.language.iso | en | |
| dc.title | 前剪接體形成之分子機制 | zh_TW |
| dc.title | Molecular Mechanism of Prespliceosome Formation | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 張典顯(Tien-Hsien Chang),吳惠南(Huey-Nan Wu),譚婉玉(Woan-Yuh Tarn),林倩伶(Chien-Ling Lin) | |
| dc.subject.keyword | 剪接反應,前剪接體,U2小核糖核酸複合體,Msl5,Prp5, | zh_TW |
| dc.subject.keyword | pre-mRNA splicing,Prespliceosome,U2 snRNP,Msl5,Prp5, | en |
| dc.relation.page | 142 | |
| dc.identifier.doi | 10.6342/NTU202001799 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-07-27 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 基因體與系統生物學學位學程 | zh_TW |
| 顯示於系所單位： | 基因體與系統生物學學位學程 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| U0001-2307202017581900.pdf 目前未授權公開取用 | 7.76 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。