- 學位論文
核醣核酸結合蛋白 Y14 參與剪接核醣小體生合成 與剪接後複合體組成之研究
The role of the RNA-binding protein Y14 in snRNP biogenesis and post-splicing complex assembly
若您是本文的作者，可授權文章由華藝線上圖書館中協助推廣。
摘要
核醣核酸（RNA）結合蛋白Y14 會和Mago 形成穩定的heterodimer，並為外顯子接合複合體（exon junction complex）當中的核心蛋白，會參與訊息核醣核酸（mRNA）的剪接（splicing）以及在細胞質裡進行的mRNA 品質管理過程中扮演重要的角色。我利用Y14/Magoh heterodimer 來尋找可能會與其產生交互作用的蛋白質，我發現Y14/Magoh 會和甲基轉移酶5（PRMT5）以及由PRMT5、pICln、MEP50 所組成的甲基轉移酶複合體（methylosome）有交互作用。我證明Y14 會促進PRMT5 的酵素活性，並形成大型的蛋白複合體。當過度表現Y14 時，PRMT5 之受質Sm 蛋白的甲基化程度有增加的情形，當Y14 的表現被抑制後，Sm 蛋白的甲基化程度會下降。Sm 蛋白為剪接核醣小體（small nuclear ribonucleoprotein, snRNP）之核心蛋白，我的研究結果指出Y14 會經由促進甲基轉移酶複合體之酵素活性，提高Sm 蛋白的甲基化程度，進而調節snRNP 在細胞質中的組合反應。 此外，我也探討Y14 之磷酸化在mRNA 剪接後的代謝過程中所扮演的角色。我發現磷酸化之後的Y14 會與mRNA 降解（mRNA decay）有關的蛋白質有較好的交互作用，包括去蓋蛋白複合體（decapping complex）、外切酶（exonuclease）、以及外切酶複合體（exosome）。另一個會與Y14/Magoh 有交互作用的PYM 蛋白也會與mRNA 降解有關的蛋白產生反應。經過實驗證實，我發現Y14 是一個cap結合蛋白，會利用其N 端蛋白區域與mRNA 的cap 結構結合。當過度表現Y14時，含有AU-rich element 之reporter 的半衰期會有增加的情形，此結果說明Y14會參與調節mRNA 的穩定性，也許是經由Y14 與cap 結構結合能力所控制。觀察細胞質內的核醣核酸降解體（processing body, P-body）是否會受到Y14 的影響時發現當Y14 的表現被抑制後，P-body 的數量會大幅下降，而當磷酸化的Y14過度表現時會促使P-body 在細胞質內聚集。我的研究結果顯示Y14 提供P-body形成與mRNA 降解之間的連結，Y14 之磷酸化可能會調節mRNA 剪接後蛋白複合體的形成以及Y14 參與mRNA 降解之反應。
並列摘要
The RNA-binding protein Y14 heterodimerizes with Mago as the core of the exon junction complex (EJC) during precursor mRNA splicing and plays a role in mRNA surveillance in the cytoplasm. Using the Y14/Magoh heterodimer as bait in a screening for its interacting partners, I identified the protein-arginine methyltransferase PRMT5 as a candidate. I show that Y14 and Magoh, but not other factors of the EJC, interact with the cytoplasmic PRMT5-containing methylosome. I further provide evidence that Y14 promoted the activity of PRMT5 in ethylation of Sm proteins of the small nuclear ibonucleoprotein (snRNP) core, whereas knockdown of Y14 reduced their methylation level. Moreover, Y14 overexpression induced the formation of a large, active, and snRNP-associated methylosome complex. However, Y14 may only transiently associate with the snRNP assembly complex in the cytoplasm. Together, my results suggest that Y14 facilitates Sm protein methylation probably by its activity in promoting the formation or stability of the methylosome-containing complex. I am currently exploring the roles of phosphorylation of Y14 in the post-splicing mRNA metabolism pathways. I found that the phosphomimetic Y14 preferred to interact with several mRNA degradation factors, including the mRNA decapping complex, decapping activators, 5’-3’ exonuclease, and the exosome-associated protein. Moreover, PYM, a partner of Y14 and Magoh, also associated with mRNA decay factors, and such associations were not dependent on Y14. In contrast, PYM competed for the interactions of mRNA decay factors with Y14. I also examined the cap-binding ability of Y14. I observed that Y14 directly bound the mRNA cap structure via its amino-terminal region and neither Magoh nor PYM abolished this interaction. The prolonged half-life of AU-rich element-containing reporter mRNA in Y14-overexpressing cells indicated that Y14 might be involved in regulation of mRNA stability. Furthermore, depletion of Y14 resulted in processing body (P-body) disappearance and overexpression of the phosphomimetic Y14 increased P-body accumulation suggesting a link between P-body formation and mRNA degradation. Taken together, I characterized the roles of Y14 in the post-splicing complexes and the activity of Y14 in mRNA decay pathway probably is regulated by its phosphorylation status.
參考文獻
1. Dreyfuss, G., Kim, V. N., and Kataoka, N. (2002) Messenger-RNA-binding proteins and the messages they carry. Nat Rev Mol Cell Biol 3, 195-205
2. Lei, E. P., and Silver, P. A. (2002) Protein and RNA export from the nucleus. Dev Cell 2, 261-272
protein MLN51 with the exon junction complex via its speckle localizer and RNA binding module. J Biol Chem 279, 33702-33715
4. Gehring, N. H., Lamprinaki, S., Kulozik, A. E., and Hentze, M. W. (2009) Disassembly of exon junction complexes by PYM. Cell 137, 536-548
5. Palacios, I. M., Gatfield, D., St Johnston, D., and Izaurralde, E. (2004) An eIF4AIII-containing complex required for mRNA localization and nonsense-mediated mRNA decay. Nature 427, 753-757
延伸閱讀
- Lee, Y. T. (2020). 利用單分子螢光共振轉移技術探討引起框架位移之RNA偽結與核醣體間交互作用 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU202003194
- Hsu, B. K. (2012). 利用系統生物學的方法研究人類如何透過微小核醣核酸和剪接因子之交亙作用以調控核醣核酸的選擇性剪接 [doctoral dissertation, National Chiao Tung University]. Airiti Library. https://doi.org/10.6842/NCTU.2012.00008
- 徐晏婷（2015）。線蟲DEAD-box核醣核酸解旋酶DDX-47及異質核醣核酸蛋白HRP-2/hnRNP Q在let-7 miRNA功能之角色〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2015.11307
- Shih, M. C. (2008). Regulation and physiological function of SF-1 binding sites on Cyp11a1 promoter [doctoral dissertation, National Tsing Hua University]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0016-0207200810155374
- Luan, C. L. (2022). A novel long noncoding RNA BCRP3 promotes protein homeostasis and cell survival by activating autophagy [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU202201725