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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Alternative splicing provides the cell the ability to generate, from a single gene, multiple protein isoforms, sometimes with different or even antagonistic functions. This process is tightly regulated and alterations in the accurate balance of alternatively spliced mRNAs are a common cause of disease. The main objective of this thesis has been to understand the molecular mechanisms underlying disease-causing defective splicing. Skipping of Fas death receptor exon 6 leads to decreased Fas-ligand induced apoptosis. We have studied how this event is promoted by a mutation at the 3’ splice site and by the proto-oncogene SF2, leading to Autoimmune Lymphoproliferative Syndrome and possibly contributing to tumor progression, respectively. Moreover, we have determined the mechanism by which an antitumor drug, Spliceostatin A, alters 3’ splice site recognition and affects alternative splicing. This thesis underscores the importance of pre-mRNA splicing in disease and how the study of disease-causing aberrant splicing can be used as a tool to understand splicing mechanisms and vice versa. / El processament alternatiu del pre-ARNm proporciona a la cèl•lula l’habilitat de generar, a partir d’un únic gen, proteïnes amb funcions diferents i, fins i tot, antagòniques. Aquest procés està altament regulat i desequilibris en l’abundància de les diferent isoformes són causes comunes de malaltia. L’objectiu principal d’aquesta tesi ha estat entendre el mecanisme molecular a través del qual problemes en el processament del pre-ARNm causen malalties. L’exclusió de l’exó 6 del receptor de mort cel•lular Fas condueix a una disminució de l’apoptosi en resposta al lligand de Fas. Hem estudiat com una mutació al lloc de processament 3’ d’aquest exó i el proto-oncogén SF2 promouen aquest patró, causant el síndrome autoimmune lifoproliferatiu i possiblement contribuint a la progressió tumorogènica, respectivament. A més, hem estudiat el mecanisme pel qual la droga antitumoral Spliceostatin A altera el reconeixement del lloc de processament 3’ i causa canvis en el processament alternatiu de diversos gens. Aquesta tesi posa en evidència la importancia del processament del pre-ARNm en malalties i com l’estudi de mutacions que alteren aquest procés i són causa de malaties pot ser utilitzat con una eina per entendre el mecanisme d’aquest processament i viceversa.

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'...

The mechanisms of pre-mRNA splicing regulation are poorly understood. Here we dissect how the Saccharomyces cerevisiae ribosomal L30 protein blocks splicing of its pre-mRNA upon binding a kink-turn structure including the 5' splice site. We show that L30 binds the nascent RPL30 transcript without preventing recognition of the 5' splice site by U1 snRNP but blocking U2 snRNP association with the branch site. Interaction of the factors BBP and Mud2p with the intron, relevant for U2 snRNP recruitment, is not affected by L30. Furthermore, the functions of neither the DEAD-box protein Sub2p in the incipient spliceosome, nor of the U2 snRNP factor Cus2p on branch site recognition, are required for L30 inhibition. These findings contrast with the effects caused by binding a heterologous protein to the same region, completely blocking intron recognition. Collectively, our data suggest that L30 represses a spliceosomal rearrangement required for U2 snRNP association with the nascent RPL30 transcript.

The informational content of 3' splice sites is low and the mechanisms whereby they are selected are not clear. Here we enunciate a set of rules that govern their selection. For many introns, secondary structures are a key factor, because they occlude alternative 3'ss from the spliceosome and reduce the effective distance between the BS and the 3'ss to a maximum of 45 nucleotides. Further alternative 3'ss are disregarded by the spliceosome because they lie at 9 nucleotides or less from the branch site, or because they are weak splice sites. With these rules, we are able to explain the splicing pattern of the vast majority of introns in Saccharomyces cerevisiae. When in excess, L30 blocks the splicing of its own transcript by interfering with a critical rearrangement that is required for the proper recognition of the intron 3' end, and thus for splicing to proceed. We show that the protein Cbp80 has a role in promoting this rearrangement and therefore antagonizes splicing regulation by L30. / Tanto la información que define el sitio de splicing 3' como los mecanismos de selección del mismo son poco conocidos. En este trabajo, proponemos una serie de reglas que gobiernan esta selección. Las estructuras secundarias son claves en el caso de muchos intrones, porque son capaces de ocultar sitios de splicing alternativos 3' al spliceosoma, y además reducen la distancia efectiva entre el punto de ramificación y el sitio de splicing 3' a un máximo de 45 nucleotidos. Otros sitios de splicing alternativo 3' no son considerados por el spliceosoma como tales porque se encuentran a 9 nucleotidos o menos del punto de ramificación, o porque son sitios de splicing débiles. Con estas reglas somos capaces de explicar el splicing de la mayoría de intrones de Saccharomyces cerevisiae. El exceso de proteína L30 bloquea el splicing de su propio tránscrito porque interfiere con la reorganización necesaria para el correcto reconocimiento del 3' final del intrón, y por tanto de su splicing. Demostramos que la proteína Cbp80 está implicada en promover esta reorganización y que por tanto antagoniza la regulación del splicing por L30.