Systemic Lupus Erythematosus (SLE) and Mixed Connective Tissue Disease (MCTD) are chronic, autoimmune disorders that target overlapping autoantigens and exhibit similar clinical manifestations. Despite 40 years of research, a reliable biomarker capable of diagnosing these syndromes has yet to be identified. Previous studies have confirmed that components of the U1 small nuclear ribonucleoprotein complex (U1 snRNP) such as U1A are 1000 fold more autoantigenic than any other nuclear component in SLE patients. Based on these findings, I hypothesize that models derived from the U1 snRNP autoantigenic properties could distinguish SLE from MCTD patients. To test this hypothesis, 30 peptides corresponding to protein regions of the U1 snRNP were tested in triplicates by indirect ELISA in sera from SLE or MCTD subjects. In addition laboratory tests and clinical manifestations data from these patients were included and analyzed in this investigation. Statistical classification methods as well as bioinformatics pattern recognition strategy were employed to determine which combination, if any, of all the variables included in this study provide the best segregation power for SLE and MCTD. The results confirmed that the IgM reactivity for U1 snRNP and U1A have the power to significantly distinguish SLE from MTCD patients as well as identify kidney and lung malfunctions for these subjects (p ≤ 0.05). Furthermore, the data analysis revealed eight novel classification rules for the segregation of SLE and MCTD which are a better classification tool than any of the currently available methods (p ≤ 0.05). Consequently, the results derived from this study support that SLE and MCTD are indeed separate disorders and pioneer the description of eight novel classification criteria capable of significantly discerning between SLE and MCTD patients (p ≤ 0.05).

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'...

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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.

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Les ARNm sont des molécules centrales pour la régulation des gènes, aidant à convertir l'information génétique stockée dans l'ADN en protéines fonctionnelles. En tant que polymère simple brin, mesurant des centaines à des milliers de nucléotides, les ARNm peuvent former des structures secondaires et tertiaires étendues formant des particules appelés ribonucléoprotéines messagères (RNPm) en s’assemblant avec des protéines. L'organisation 3D des (pré-)RNPm influence de nombreux aspects de leur métabolisme, incluant la régulation de leur maturation, de leur export et de leur traduction dans le cytoplasme. Malgré leur importance, notre compréhension de l'organisation structurelle des (pré-)RNPm in vivo, et des principes qui la régissent est minime. Au cours de ma thèse, j'ai analysé l'organisation des (pré-)mRNP en développant une vision centrée sur l'ARN. Pour cela, j'ai mis en place une approche combinant l'hybridation in situ d'ARN monomoléculaire (smFISH) avec la microscopie à illumination structurée (SIM) et l'ai utilisée pour étudier l'organisation des mRNP dans le noyau et le cytoplasme. Nos résultats suggèrent que l'organisation (pré-)mRNP varie à différents stades de sa vie. Nous montrons que l'empaquetage (pré-)mRNP commence de manière co-transcriptionnelle, avec des introns organisés en conformations compactes. Cette organisation est modifiée au cours de la transcription au fur et à mesure que la polymérase se déplace le long du gène, assemblant finalement un intron avec les extrémités à proximité l’une de l’autre, d'une manière dépendante du spliceosome, suggérant que l'organisation co-transcriptionnelle des introns pourrait être critique pour déterminer son excision. Une fois libérés, les mRNP ont une organisation linéaire compacte dans le nucléoplasme et éventuellement une conformation en tige. L'organisation d’un mRNP dans le cytoplasme est influencée par sa traduction. Alors que la traduction ouvre les mRNP, la séparation des extrémités de l'ARNm, l'inhibition de la traduction et la libération de ribosomes, ou le recrutement dans les granules de stress, donnent aux mRNP une structure très compacte. Fait intéressant, nous trouvons rarement des ARNm avec les extrémités 5' et 3' à proximité, ce qui suggère que la traduction en boucle fermée n'est pas un état universel pour tous les ARNm en cours de traduction. Ensemble, nos résultats fournissent une image essentielle de l'organisation du mRNP dans les cellules et souligne le rôle important de la conformation du RNPm dans la régulation de la traduction et de la maturation d’une RNPm. / mRNAs act as the central molecules in gene regulation, helping convert the genetic information stored in the DNA to functional proteins. As a single-stranded polymer, hundreds to thousands of nucleotides in length, mRNAs can form extensive secondary and tertiary structures and, together with proteins, are packaged into assemblies called messenger ribonucleoproteins (mRNPs). The 3D organisation of (pre-)mRNPs influences many aspects of what happens to them, including regulating their processing, export and translation in the cytoplasm. Despite their significance, our understanding of the structural organisation of (pre-)mRNPs in vivo is minimal, as is our comprehension of the principles that govern it. During my PhD, I have developed an RNA-centric view on (pre-)mRNP organisation. For this, I have established an approach combining single-molecule RNA in situ hybridisation (smFISH) with structured illumination microscopy (SIM) and used it to study mRNP organisation in the nucleus and cytoplasm. Our results suggest that (pre-)mRNP organisation is altered at various stages during its lifetime. We show that (pre-)mRNP packaging starts co-transcriptionally, with introns organised into compact conformations. This organisation is altered during the course of transcription as the polymerase travels along the gene, finally assembling an intron with the ends in proximity in a spliceosome dependent manner, suggesting that co-transcriptional intron organisation could be critical in determining its excision. Once released, mRNPs have a compact linear organisation in the nucleoplasm and possibly a rod-like conformation. mRNP organisation in the cytoplasm is influenced by its translational status. While translation opens up mRNPs, separating the ends of the mRNA, translation inhibition and release of ribosomes, or recruitment to stress granules result in mRNPs having a highly compact structure. Interestingly, we rarely find mRNAs with the 5’ and 3’ ends in proximity, suggesting that closed-looped translation is not a universal state for all translating mRNAs. Together, our results provide a unique and essential view of mRNP organisation in cells and reveal important insight into the role of mRNP conformation in regulating translation and mRNP processing.