臺灣博碩士論文加值系統

RNA激活 (RNA activation, RNAa) 是一種重要的基因表達調控方式，它能透過促進目標基因轉錄的進行，上調特定基因的表達，這對研究基因功能以及治療基因缺陷相關的疾病上有著極高的價值。RNAa的作用機制雖與RNA干擾 （RNA interference, RNAi）類似，但涉及的功能性蛋白稍有不同，且與RNAi中的基因沉默現象相反，RNAa能夠對基因表達進行正向調控，繼而影響細胞的功能性與適應性。

目前已知RNAa的核心複合體RITA (RNA-induced transcriptional activation complex)主要由AGO2、saRNA (small activating RNA)和RHA (RNA helicase A)組成。其中saRNA-AGO2負責引導RITA複合體至目標基因，RHA繼而促進基因的激活，三者各自具備重要的功能。因此，深入了解它們如何相互作用以形成功能完整的RITA複合體，將能為之後RNAa的研究建立起良好的基礎。

在本研究中，我利用蛋白質過表達的方法，在細胞內分析了AGO2和RHA之間的結合關係，發現了兩者的最小結合域，從而更清晰地揭示了RITA複合體的形成機制。此外，我藉由對RNAa分子機制的理解，利用生物工程將AGO2和RHA的主要功能區域製作成融合蛋白，並通過多項測試驗證其功效，顯示RNAa具有廣泛的應用價值。隨著對RNAa機制的進一步深入研究，未來還將開發出更多不同的應用方式。

RNA activation (RNAa) is an important mechanism for regulating gene expression. It involves the promotion of target gene transcription, resulting in the upregulation of specific gene expression. This process holds great value in the study of gene function and the treatment of diseases related to gene defects. While the mechanism of RNAa is similar to RNA interference (RNAi), it involves slightly different functional proteins. Unlike the gene silencing phenomenon in RNAi, RNAa can positively regulate gene expression, thereby influencing the functionality and adaptability of cells.

The RITA (RNA-induced transcriptional activation complex) of RNA activation (RNAa) is known to be primarily composed of AGO2, saRNA, and RHA. The saRNA-AGO2 guides the RITA complex to the target gene, while RHA facilitates gene activation. Each component possesses crucial functionalities. Therefore, understanding of their interactions to form a fully functional RITA complex will establish a solid foundation for future research in RNAa.

In this study, I used protein overexpression methods to analyze the binding relationship between AGO2 and RHA in cells and discovered their minimum binding domain, thereby revealing the mechanism of RITA complex formation more clearly. Additionally, based on RNAa, I bioengineered the main functional domains of AGO2 and RHA into fusion proteins and validated their efficacy through multiple tests, demonstrating the broad application potential of RNAa. With further in-depth research on the mechanism of RNAa, more diverse applications will be developed in the future.

致謝 i
中文摘要 ii
英文摘要 iii
目錄 iv
圖目錄 vi
第一章 緒論 1
1.1 RNA干擾路徑 (RNA interference pathway) 1
1.1.1小片段RNA (small RNA, sRNA) 2
1.1.2 Argonaute蛋白 (AGO) 3
1.2 RNA激活路徑 (RNA activation pathway) 4
1.2.1 RNA誘導轉錄活化複合體 (RITA) 6
1.2.2 RHA蛋白 (RNA helicase A) 7
1.2.3 RNAa潛在的應用價值 8
1.3 研究動機與目標 9
第二章 實驗結果 10
2.1 RHA與AGO2的交互作用 10
2.2 RHA (265-1270)與AGO2的交互作用 12
2.3 RHA (265-830)與AGO2 (1-447)為RITA內的最小結合區域 14
2.4 AGO2具有轉錄活化的能力 18
2.5 NLS-AD-AGO2活化抑癌基因p21 20
2.6 RNA-seq / ChIP-seq挑選出細胞內受RNAa所調控的內源性基因 23
2.7 NLS-AD-AGO2活化受RNAa所調控的內源性基因 25
第三章 討論 28
第四章 實驗方法 33
4.1質體構築 33
4.2重組蛋白在人類細胞的表現與分析 35
4.3自動誘導蛋白表現系統 (Autoinduction of protein expression system) 38
4.4蛋白與蛋白間結合能力之分析 (Pull-down assay) 38
4.5冷光報告基因活性檢測 (Luciferase assay) 39
4.6定量反轉錄PCR (RT-qPCR) 39
4.7染色質免疫沉澱 (Chromatin Immunoprecipitation, ChIP) 40
第五章 參考文獻 42
第六章 附錄 47

Aratani, S., Fujii, R., Oishi, T., Fujita, H., Amano, T., Ohshima, T., Hagiwara, M., Fukamizu, A., & Nakajima, T. (2001). Dual roles of RNA helicase A in CREB-dependent transcription. Mol Cell Biol, 21(14), 4460-4469. https://doi.org/10.1128/mcb.21.14.4460-4469.2001
Aratani, S., Oishi, T., Fujita, H., Nakazawa, M., Fujii, R., Imamoto, N., Yoneda, Y., Fukamizu, A., & Nakajima, T. (2006). The nuclear import of RNA helicase A is mediated by importin-alpha3. Biochem Biophys Res Commun, 340(1), 125-133. https://doi.org/10.1016/j.bbrc.2005.11.161
Chu, Y., Simic, R., Warner, M. H., Arndt, K. M., & Prelich, G. (2007). Regulation of histone modification and cryptic transcription by the Bur1 and Paf1 complexes. Embo j, 26(22), 4646-4656. https://doi.org/10.1038/sj.emboj.7601887
Chu, Y., Yue, X., Younger, S. T., Janowski, B. A., & Corey, D. R. (2010). Involvement of argonaute proteins in gene silencing and activation by RNAs complementary to a non-coding transcript at the progesterone receptor promoter. Nucleic Acids Res, 38(21), 7736-7748. https://doi.org/10.1093/nar/gkq648
Darnell, J. C., Jensen, K. B., Jin, P., Brown, V., Warren, S. T., & Darnell, R. B. (2001). Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function. Cell, 107(4), 489-499. https://doi.org/10.1016/s0092-8674(01)00566-9
Diederichs, S., & Haber, D. A. (2007). Dual role for argonautes in microRNA processing and posttranscriptional regulation of microRNA expression. Cell, 131(6), 1097-1108. https://doi.org/10.1016/j.cell.2007.10.032
Fidaleo, M., De Paola, E., & Paronetto, M. P. (2016). The RNA helicase A in malignant transformation. Oncotarget, 7(19), 28711-28723. https://doi.org/10.18632/oncotarget.7377
Fu, Q., & Yuan, Y. A. (2013). Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9). Nucleic Acids Res, 41(5), 3457-3470. https://doi.org/10.1093/nar/gkt042
Gebert, L. F. R., & MacRae, I. J. (2019). Regulation of microRNA function in animals. Nat Rev Mol Cell Biol, 20(1), 21-37. https://doi.org/10.1038/s41580-018-0045-7
Ghisolfi, L., Kharrat, A., Joseph, G., Amalric, F., & Erard, M. (1992). Concerted activities of the RNA recognition and the glycine-rich C-terminal domains of nucleolin are required for efficient complex formation with pre-ribosomal RNA. Eur J Biochem, 209(2), 541-548. https://doi.org/10.1111/j.1432-1033.1992.tb17318.x
Giorgi, C., Cogoni, C., & Catalanotto, C. (2012). From transcription to translation: new insights in the structure and function of Argonaute protein. Biomol Concepts, 3(6), 545-559. https://doi.org/10.1515/bmc-2012-0024
Hartman, T. R., Qian, S., Bolinger, C., Fernandez, S., Schoenberg, D. R., & Boris-Lawrie, K. (2006). RNA helicase A is necessary for translation of selected messenger RNAs. Nat Struct Mol Biol, 13(6), 509-516. https://doi.org/10.1038/nsmb1092
Hu, J., Chen, Z., Xia, D., Wu, J., Xu, H., & Ye, Z. Q. (2012). Promoter-associated small double-stranded RNA interacts with heterogeneous nuclear ribonucleoprotein A2/B1 to induce transcriptional activation. Biochem J, 447(3), 407-416. https://doi.org/10.1042/bj20120256
Jaehning, J. A. (2010). The Paf1 complex: platform or player in RNA polymerase II transcription? Biochim Biophys Acta, 1799(5-6), 379-388. https://doi.org/10.1016/j.bbagrm.2010.01.001
Janowski, B. A., Younger, S. T., Hardy, D. B., Ram, R., Huffman, K. E., & Corey, D. R. (2007). Activating gene expression in mammalian cells with promoter-targeted duplex RNAs. Nat Chem Biol, 3(3), 166-173. https://doi.org/10.1038/nchembio860
Kiledjian, M., & Dreyfuss, G. (1992). Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box. Embo j, 11(7), 2655-2664. https://doi.org/10.1002/j.1460-2075.1992.tb05331.x
Kim, B., Jeong, K., & Kim, V. N. (2017). Genome-wide Mapping of DROSHA Cleavage Sites on Primary MicroRNAs and Noncanonical Substrates. Mol Cell, 66(2), 258-269.e255. https://doi.org/10.1016/j.molcel.2017.03.013
Kim, J., Guermah, M., & Roeder, R. G. (2010). The human PAF1 complex acts in chromatin transcription elongation both independently and cooperatively with SII/TFIIS. Cell, 140(4), 491-503. https://doi.org/10.1016/j.cell.2009.12.050
Krogan, N. J., Kim, M., Ahn, S. H., Zhong, G., Kobor, M. S., Cagney, G., Emili, A., Shilatifard, A., Buratowski, S., & Greenblatt, J. F. (2002). RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach. Mol Cell Biol, 22(20), 6979-6992. https://doi.org/10.1128/mcb.22.20.6979-6992.2002
Kwon, S. C., Nguyen, T. A., Choi, Y. G., Jo, M. H., Hohng, S., Kim, V. N., & Woo, J. S. (2016). Structure of Human DROSHA. Cell, 164(1-2), 81-90. https://doi.org/10.1016/j.cell.2015.12.019
Lee, C. G., Eki, T., Okumura, K., Nogami, M., Soares Vda, C., Murakami, Y., Hanaoka, F., & Hurwitz, J. (1999). The human RNA helicase A (DDX9) gene maps to the prostate cancer susceptibility locus at chromosome band 1q25 and its pseudogene (DDX9P) to 13q22, respectively. Somat Cell Mol Genet, 25(1), 33-39. https://doi.org/10.1023/b:scam.0000007138.44216.3a
Li, L. C. (2017). Small RNA-Guided Transcriptional Gene Activation (RNAa) in Mammalian Cells. Adv Exp Med Biol, 983, 1-20. https://doi.org/10.1007/978-981-10-4310-9_1
Li, L. C., Okino, S. T., Zhao, H., Pookot, D., Place, R. F., Urakami, S., Enokida, H., & Dahiya, R. (2006). Small dsRNAs induce transcriptional activation in human cells. Proc Natl Acad Sci U S A, 103(46), 17337-17342. https://doi.org/10.1073/pnas.0607015103
Liu, H., Lei, C., He, Q., Pan, Z., Xiao, D., & Tao, Y. (2018). Nuclear functions of mammalian MicroRNAs in gene regulation, immunity and cancer. Mol Cancer, 17(1), 64. https://doi.org/10.1186/s12943-018-0765-5
Liu, J., Carmell, M. A., Rivas, F. V., Marsden, C. G., Thomson, J. M., Song, J. J., Hammond, S. M., Joshua-Tor, L., & Hannon, G. J. (2004). Argonaute2 is the catalytic engine of mammalian RNAi. Science, 305(5689), 1437-1441. https://doi.org/10.1126/science.1102513
MacRae, I. J., Zhou, K., & Doudna, J. A. (2007). Structural determinants of RNA recognition and cleavage by Dicer. Nat Struct Mol Biol, 14(10), 934-940. https://doi.org/10.1038/nsmb1293
Marton, H. A., & Desiderio, S. (2008). The Paf1 complex promotes displacement of histones upon rapid induction of transcription by RNA polymerase II. BMC Mol Biol, 9, 4. https://doi.org/10.1186/1471-2199-9-4
Matranga, C., Tomari, Y., Shin, C., Bartel, D. P., & Zamore, P. D. (2005). Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell, 123(4), 607-620. https://doi.org/10.1016/j.cell.2005.08.044
Mueller, C. L., & Jaehning, J. A. (2002). Ctr9, Rtf1, and Leo1 are components of the Paf1/RNA polymerase II complex. Mol Cell Biol, 22(7), 1971-1980. https://doi.org/10.1128/mcb.22.7.1971-1980.2002
Nakajima, T., Uchida, C., Anderson, S. F., Lee, C. G., Hurwitz, J., Parvin, J. D., & Montminy, M. (1997). RNA helicase A mediates association of CBP with RNA polymerase II. Cell, 90(6), 1107-1112. https://doi.org/10.1016/s0092-8674(00)80376-1
Nakanishi, K., Weinberg, D. E., Bartel, D. P., & Patel, D. J. (2012). Structure of yeast Argonaute with guide RNA. Nature, 486(7403), 368-374. https://doi.org/10.1038/nature11211
Neilsen, C. T., Goodall, G. J., & Bracken, C. P. (2012). IsomiRs--the overlooked repertoire in the dynamic microRNAome. Trends Genet, 28(11), 544-549. https://doi.org/10.1016/j.tig.2012.07.005
Nguyen, T. A., Jo, M. H., Choi, Y. G., Park, J., Kwon, S. C., Hohng, S., Kim, V. N., & Woo, J. S. (2015). Functional Anatomy of the Human Microprocessor. Cell, 161(6), 1374-1387. https://doi.org/10.1016/j.cell.2015.05.010
Okada, C., Yamashita, E., Lee, S. J., Shibata, S., Katahira, J., Nakagawa, A., Yoneda, Y., & Tsukihara, T. (2009). A high-resolution structure of the pre-microRNA nuclear export machinery. Science, 326(5957), 1275-1279. https://doi.org/10.1126/science.1178705
Park, J. E., Heo, I., Tian, Y., Simanshu, D. K., Chang, H., Jee, D., Patel, D. J., & Kim, V. N. (2011). Dicer recognizes the 5' end of RNA for efficient and accurate processing. Nature, 475(7355), 201-205. https://doi.org/10.1038/nature10198
Place, R. F., Noonan, E. J., Földes-Papp, Z., & Li, L. C. (2010). Defining features and exploring chemical modifications to manipulate RNAa activity. Curr Pharm Biotechnol, 11(5), 518-526. https://doi.org/10.2174/138920110791591463
Place, R. F., Wang, J., Noonan, E. J., Meyers, R., Manoharan, M., Charisse, K., Duncan, R., Huang, V., Wang, X., & Li, L. C. (2012). Formulation of Small Activating RNA Into Lipidoid Nanoparticles Inhibits Xenograft Prostate Tumor Growth by Inducing p21 Expression. Mol Ther Nucleic Acids, 1(3), e15. https://doi.org/10.1038/mtna.2012.5
Rand, T. A., Petersen, S., Du, F., & Wang, X. (2005). Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell, 123(4), 621-629. https://doi.org/10.1016/j.cell.2005.10.020
Ranji, A., Shkriabai, N., Kvaratskhelia, M., Musier-Forsyth, K., & Boris-Lawrie, K. (2011). Features of double-stranded RNA-binding domains of RNA helicase A are necessary for selective recognition and translation of complex mRNAs. J Biol Chem, 286(7), 5328-5337. https://doi.org/10.1074/jbc.M110.176339
Rondón, A. G., Gallardo, M., García-Rubio, M., & Aguilera, A. (2004). Molecular evidence indicating that the yeast PAF complex is required for transcription elongation. EMBO Rep, 5(1), 47-53. https://doi.org/10.1038/sj.embor.7400045
Schirle, N. T., & MacRae, I. J. (2012). The crystal structure of human Argonaute2. Science, 336(6084), 1037-1040. https://doi.org/10.1126/science.1221551
Schlegel, B. P., Starita, L. M., & Parvin, J. D. (2003). Overexpression of a protein fragment of RNA helicase A causes inhibition of endogenous BRCA1 function and defects in ploidy and cytokinesis in mammary epithelial cells. Oncogene, 22(7), 983-991. https://doi.org/10.1038/sj.onc.1206195
Schwartz, J. C., Younger, S. T., Nguyen, N. B., Hardy, D. B., Monia, B. P., Corey, D. R., & Janowski, B. A. (2008). Antisense transcripts are targets for activating small RNAs. Nat Struct Mol Biol, 15(8), 842-848. https://doi.org/10.1038/nsmb.1444
Tang, W., You, W., Shi, F., Qi, T., Wang, L., Djouder, Z., Liu, W., & Zeng, X. (2009). RNA helicase A acts as a bridging factor linking nuclear beta-actin with RNA polymerase II. Biochem J, 420(3), 421-428. https://doi.org/10.1042/bj20090402
Tian, Y., Simanshu, D. K., Ma, J. B., Park, J. E., Heo, I., Kim, V. N., & Patel, D. J. (2014). A phosphate-binding pocket within the platform-PAZ-connector helix cassette of human Dicer. Mol Cell, 53(4), 606-616. https://doi.org/10.1016/j.molcel.2014.01.003
Tsutsumi, A., Kawamata, T., Izumi, N., Seitz, H., & Tomari, Y. (2011). Recognition of the pre-miRNA structure by Drosophila Dicer-1. Nat Struct Mol Biol, 18(10), 1153-1158. https://doi.org/10.1038/nsmb.2125
Turunen, M. P., Lehtola, T., Heinonen, S. E., Assefa, G. S., Korpisalo, P., Girnary, R., Glass, C. K., Väisänen, S., & Ylä-Herttuala, S. (2009). Efficient regulation of VEGF expression by promoter-targeted lentiviral shRNAs based on epigenetic mechanism: a novel example of epigenetherapy. Circ Res, 105(6), 604-609. https://doi.org/10.1161/circresaha.109.200774
Wang, X., Wang, J., Huang, V., Place, R. F., & Li, L. C. (2012). Induction of NANOG expression by targeting promoter sequence with small activating RNA antagonizes retinoic acid-induced differentiation. Biochem J, 443(3), 821-828. https://doi.org/10.1042/bj20111491
Wang, Y., Sheng, G., Juranek, S., Tuschl, T., & Patel, D. J. (2008). Structure of the guide-strand-containing argonaute silencing complex. Nature, 456(7219), 209-213. https://doi.org/10.1038/nature07315
Wilson, R., Ainscough, R., Anderson, K., Baynes, C., Berks, M., Bonfield, J., Burton, J., Connell, M., Copsey, T., Cooper, J., & et al. (1994). 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature, 368(6466), 32-38. https://doi.org/10.1038/368032a0
Yue, X., Schwartz, J. C., Chu, Y., Younger, S. T., Gagnon, K. T., Elbashir, S., Janowski, B. A., & Corey, D. R. (2010). Transcriptional regulation by small RNAs at sequences downstream from 3' gene termini. Nat Chem Biol, 6(8), 621-629. https://doi.org/10.1038/nchembio.400
Zamore, P. D., Tuschl, T., Sharp, P. A., & Bartel, D. P. (2000). RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell, 101(1), 25-33. https://doi.org/10.1016/s0092-8674(00)80620-0
Zhang, S., & Grosse, F. (1994). Nuclear DNA helicase II unwinds both DNA and RNA. Biochemistry, 33(13), 3906-3912. https://doi.org/10.1021/bi00179a016
Zhang, S., & Grosse, F. (1997). Domain structure of human nuclear DNA helicase II (RNA helicase A). J Biol Chem, 272(17), 11487-11494. https://doi.org/10.1074/jbc.272.17.11487
Zhang, S., Schlott, B., Görlach, M., & Grosse, F. (2004). DNA-dependent protein kinase (DNA-PK) phosphorylates nuclear DNA helicase II/RNA helicase A and hnRNP proteins in an RNA-dependent manner. Nucleic Acids Res, 32(1), 1-10. https://doi.org/10.1093/nar/gkg933
Zhang, Y., Sikes, M. L., Beyer, A. L., & Schneider, D. A. (2009). The Paf1 complex is required for efficient transcription elongation by RNA polymerase I. Proc Natl Acad Sci U S A, 106(7), 2153-2158. https://doi.org/10.1073/pnas.0812939106
Liu, Z., Johnson, S. T., Zhang, Z., & Corey, D. R. (2019). Expression of TNRC6 (GW182)
Proteins Is Not Necessary for Gene Silencing by Fully Complementary RNA Duplexes. Nucleic Acid Ther, 29(6), 323-334. https://doi.org/10.1089/nat.2019.0815
Ebert, M. S., & Sharp, P. A. (2010). MicroRNA sponges: progress and possibilities. Rna, 16(11), 2043-2050. https://doi.org/10.1261/rna.2414110