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Pre-mRNA splicing is a process in which introns are removed from eukaryotic transcripts and exons are ligated together. Splicing is catalyzed by spliceosome, a large ribonucleoprotein complex composed of five small nuclear RNAs and more than 100 additional proteins, which recognizes 5' splice site, branch point site and 3' splice site and performs two transesterification reactions to produce mRNA molecules. 5' splice site is recognized by U1 snRNP and U2 auxiliary factor (U2AF) is involved in branch point and 3' splice site recognition in the early splicing complex. There is some evidence of splice sites cooperation during intron recognition in vitro but little is known about the situation in vivo. Using Fluorescence resonance energy transfer (FRET) and RNA immunoprecipitation (RIP) methods, we have investigated the early stages of spliceosome assembly. We have employed splicing reporters based on -globin gene and MS2 stem loops to detect interactions of proteins on RNA molecule directly in the cell nucleus. Results of FRET indicate that intact 5' splice site is required for U2AF35 interaction with 3' splice site and that U1C recruitment to 5' splice site is partially limited upon 3' splice site mutation. We have also confirmed by RIP that U2 snRNP association with pre-mRNA molecule requires presence of 5'...

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24268号 / 生博第482号 / 新制||生||64(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 片山 高嶺, 教授 石川 冬木, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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hPrp8 is an essential pre-mRNA splicing factor. This highly conserved protein is a component of the U5 small ribonucleoprotein particle (U5 snRNP), which constitutes one of the building blocks of the spliceosome. hPrp8 acts as a key regulator of spliceosome activation and interacts directly with U5 snRNA and with the regions of pre-mRNA that are involved in the transesterification reactions during splicing. Mutations in hPrp8 have been shown to cause an autosomal dominant form of retinitis pigmentosa (RP), an inherited disease leading to progressive degeneration of retina. In this study, we analyzed the effects of the RP-associated mutations on the function of hPrp8. Using BAC recombineering, we created mutant variants of hPrp8-GFP construct and we generated stable cell lines expressing the recombinant proteins. The mutant proteins were expressed and localized to the nucleus. However, one of the missense mutations affected the localization and stability of hPrp8. Further experiments suggested that RP-associated mutations affect the ability of hPrp8 to interact with other components of the U5 snRNP and with pre-mRNA. We further studied the biogenesis of U5 snRNP. We depleted hPrp8 by siRNA to interfere with U5 snRNP assembly and we observed that the incompletely assembled U5 snRNPs accumulate in...

Regulation of transcription elongation by P-TEFb is critical for proper gene expression and cell survival. The cell possesses large quantities of P-TEFb, but the vast majority of it is locked away and inactive in the 7SK snRNP. Since the discovery of the 7SK snRNP, research has been conducted to determine how P-TEFb is released from this complex. The goal of the research presented in this thesis is to better understand how the 7SK snRNP regulates P-TEFb and ultimately, gene expression. This work documents the discovery and characterization of the 7SK stability protein LARP7. LARP7 is is associated with 7SK regardless of the presence of P-TEFb and HEXIM1. Stabilization of 7SK is essential for maintenance of the RNP because loss of LARP7 results in an increase in free P-TEFb and a significant reduction in the amount of 7SK. These results indicate that stabilization of the 7SK snRNP by LARP7 is important for regulating P-TEFb homeostasis. Although P-TEFb was first characterized from Drosophila lysates, the conservation of the 7SK snRNP and the mechanisms regulating P-TEFb inhibition have not been described. Here, the Drosophila melanogaster homologues of LARP7 and 7SK are characterized. These studies show that the system of P-TEFb regulation is similar in flies and this makes Drosophila an attractive model system for studying P-TEFb regulation through embryonic and larval development. Finally, factors and modifications involved in releasing P-TEFb directly are explored. An assay was developed for discovering proteins that can bind to and release P-TEFb from the 7SK snRNP. Use of this assay showed that post-translational modification of the components of the 7SK snRNP do not cause P-TEFb release directly. However, HIV Tat and the C-terminal P-TEFb binding region of the bromodomain containing protein, Brd4, are capable of extracting P-TEFb directly. Most importantly, the release of P-TEFb is followed by a conformational change in 7SK RNA that prevents the continued binding of HEXIM1 to the complex. P-TEFb release from the 7SK snRNP is the result of direct extraction of P-TEFb by viral or cellular proteins, and not post-translational modifications or a competition between HEXIM1 and hnRNP proteins for 7SK binding.