Trabalhos recentes indicam que a maior parte do transcriptoma de células de mamíferos é composto por RNAs não-codificadores de proteínas (ncRNAs). Nosso grupo tem identificado e caracterizado ncRNAs longos (>200 nt), sem splicing, expressos em regiões intrônicas de genes codificadores de proteína. Contudo, a biogênese, processamento e localização sub-celular desta classe de RNAs permanecem desconhecidos. Este trabalho teve como objetivos i) investigar a contribuição da RNA Polimerase II (RNAP II) na transcrição de ncRNAs intrônicos, ii) avaliar a meia-vida destes ncRNAs em relação a mRNAs, e iii) verificar a distribuição sub-celular de ncRNAs intrônicos. Os resultados obtidos indicaram que ncRNAs intrônicos são predominantemente transcritos pela RNAP II a partir de regiões promotoras funcionalmente semelhantes as que controlam a transcrição de mRNAs. Ensaios de estabilidade revelaram que, em média, ncRNAs intrônicos possuem meia-vida igual ou maior (3,4h a 4,2h) do que mRNAs (3,1h). A maior parte dos ncRNAs intrônicos possui estrutura cap 5\', sugerindo que sejam estabilizados para desempenhar papéis na biologia da célula que não dependam de um rápido turnover. A maior parte dos ncRNAs intrônicos é exportada para o citoplasma, indicando que devam exercer alguma função biológica neste compartimento. Em conjunto, este trabalho fornece informações novas a respeito da biogênese, estabilidade e localização sub-celular ncRNAs intrônicos expressos em células humanas, contribuindo para avançar o conhecimento sobre esta classe de transcritos celulares. / Recent studies have shown that most of the mammalian transcriptome is comprised of non-coding RNAs (lncRNAs). Our group has identified and characterized long (>200 nt), unspliced lncRNAs expressed in intronic regions of protein coding genes. However, the biogenesis, processing, stability and subcellular localization of members from this RNA class remain unknown. The aims of this work were i) to investigate the contribution of RNA Polymerase II (RNAP II) to the transcription of intronic, ii) to evaluate the half-life of these ncRNAs relative to mRNAs, and iii) determine their subcellular distribution. Our results indicate that intronic ncRNAs are predominantly transcribed by RNAP II from promoter regions functionally similar to those that control the transcription of mRNAs. Stability assays revealed that intronic ncRNAs have an average half-life equal or greater (3.4h to 4.2h) than mRNAs (3.1h). The majority of intronic ncRNAs have 5\' cap modification suggesting that these transcripts are stabilized, possibly to exert roles in the biology of the cell that does not depend on a rapid turnover. Although intronic ncRNAs do not encode proteins, most of these transcripts are transported to the cytoplasm which indicates that they may perform some biological function in this compartment. Altogether, this study reveals with novel information regarding the biogenesis, stability and subcellular localization of intronic ncRNAs expressed in human cells, thus contributing to advance the knowledge on this class of cellular transcripts.

No description available.

Die Genexpressionsmaschinerie in Chloroplasten und Mitochondrien und die ihrer prokaryotischen Vorläufer sind konserviert. Innerhalb eines bakteriellen Grundgerüsts entwickelte sich darüber hinaus ein komplexer RNA-Metabolismus. In der vorliegenden Arbeit wird eine neue Klasse kleiner RNAs (15-50nt) mit plastidärem und mitochondrialen Ursprung beschrieben. Diese kurzen RNAs überlappen mit Bindestellen von RNA-bindenden Proteinen, die mRNAs gegen exonukleolytischen Verdau beschützen. Diese stabilisierende Funktion wird vermutlich hauptsächlich von PPR (Pentatricopeptid repeat) Proteinen und verwandten Proteine bewerkstelligt. Die kleinen RNAs repräsentieren dabei minimale nuklease-resistente Bereiche, sogenannte RNA-Bindeprotein footprints. Solche footprints finden sich in fast jedem intergenischen Bereich, der Prozessierung aufweist. Durch transkriptomweite Untersuchungen von kleinen RNAs in Mutanten von RNA-Bindeproteinen konnte für diese eine Reihe von Bindestellen identifiziert werden. Nuklease-resistente kleine RNAs fehlen in entsprechenden Mutanten. Der Vergleich neu identifizierter Ziele einzelner RNA-Bindeproteine führte dabei zu neuen Erkenntnissen über den Mechanismus der RNA-Erkennung durch PPR Proteine. Im Gegensatz zu Plastiden befinden sich kleine RNAs in Mitochondrien überwiegend an den 3‘ Enden von Transkripten, deren Stabilität vermutlich maßgeblich von diesen RNA-Bindeproteinen beeinflusst wird. Für das chloroplastidäre Ribonukleoprotein CP31A konnte gezeigt werden, dass es an der Stabilisierung der ndhF mRNA beteiligt ist. Die Interaktion mit der ndhF mRNA, die eine zentrale Komponente des NDH-Komplexes kodiert, wird dabei über die 3‘ untranslatierte Region vermittelt. Zusätzlich konnte gezeigt werden, dass CP31A die Stabilität einiger antisense Transkripte beeinflusst. Weiterhin wurden zehn neue Cytidin Desaminierungungen durch die Analyse von RNA-Seq Datensätzen in der Modellpflanze Arabidopsis thaliana identifiziert. / Chloroplasts and mitochondria are of endosymbiotic origin. Their basic gene expression machineries are retained from their free-living prokaryotic progenitors. On top of this bacterial scaffold, a number of organelle-specific RNA processing steps evolved. In this thesis, a novel class of organelle-specific short (15-50nt) RNAs is described on a transcriptome-wide scale. The small RNAs are found at binding sites of PPR (Pentatricopeptide repeat) and PPR-like proteins, which protect mRNAs against exonucleolytic decay. The small RNAs represent minimal nuclease resistant RNAs, so called PPR footprints. Small RNAs were identified in almost every intergenic region subjected to intergenic processing. This finding suggests that accumulation of processed transcripts in plastids is mostly due to protection by highly specific RNA-binding proteins. Small RNA sequencing identified a number of nuclease insensitive sites missing in mutants of RNA-binding proteins. Analysis of multiple small RNAs representing target sites of single PPR proteins expands the knowledge of target specificity. In mitochondria, accumulations of small RNAs predicts that at least two thirds of mitochondrial mRNAs are stabilized by RNA-binding proteins binding in their 3’UTR. In sum, small organellar RNAs turned out to be instrumental in elucidating the hitherto enigmatic intercistronic processing of organellar RNAs and allowed novel insights into the function of the dominant family of organellar RNA binding proteins, the PPR proteins. A chloroplast ribonucleoprotein CP31A is shown to be involved in stabilization of an mRNA for a central component of the NDH-complex by interaction with its 3’UTR. In addition, CP31A represents the first factor described that influences the accumulation of chloroplast antisense transcripts. Finally, ten novel plastid C to U RNA-editing sites were identified in the model plant Arabidopsis thaliana, using a novel RNA-Seq based approach.

mRNA degradation is a fundamental process that controls both the level and the fidelity of gene expression. Using a combination of bioinformatic, genomic, genetic, and molecular biology approaches, we have shown that Edc3p, a yeast mRNA decay factor, controls the stability of the intron-containing YRA1 pre-mRNA. We found that Edc3p-mediated degradation of YRA1 pre-mRNA: 1) is a component of a negative feedback loop involved in the autoregulation of YRA1, 2) takes place in the cytoplasm, 3) is independent of translation, 4) occurs through a deadenylation-independent decapping and 5΄ to 3΄ exonucleotic decay mechanism, and 5) is controlled by specific cis-acting elements and trans-regulatory factors. Cis-regulation of YRA1 pre-mRNA degradation is complicated and precise. Sequences in exon1 inhibit YRA1 pre-mRNA splicing and/or promote pre-mRNA export in a size-dependent but sequence-independent manner. Sequences in the intron dictate the substrate specificity for Edc3p-mediated decay. Five structurally different but functionally interdependent modules were identified in the YRA1 intron. Two modules, designated Edc3p-responsive elements (EREs), are required for triggering an Edc3p-response. Three other modules, designated translational repression elements (TREs), are required for repressing translation of YRA1 pre-mRNA. TREs enhance the efficiency of the response of the EREs to Edc3p by inhibiting translation-dependent nonsense-mediated mRNA decay (NMD). Trans-regulation of YRA1 pre-mRNA is governed by Yra1p, which inhibits YRA1 pre-mRNA splicing and commits the pre-mRNA to nuclear export, and the RNP export factors, Mex67p and Crm1p, which jointly promote YRA1 pre-mRNA export. Mex67p also appears to interact with sequences in the YRA1 intron to promote translational repression and to enhance the Edc3p response of YRA1 pre-mRNA. These results illustrate how common steps in the nuclear processing, export, and degradation of a transcript can be uniquely combined to control the expression of a specific gene and suggest that Edc3p-mediated decay may have additional regulatory functions in eukaryotic cells.

No description available.

Heterozygous germline defects in a gene encoding a type II receptor for bone morphogenetic proteins (BMPR-II) underlie the majority of inherited cases of the vascular disorder known as pulmonary arterial hypertension (PAH). However, the precise molecular consequences of PAH causing mutations on the function of the receptor complex remain unclear. We employed novel enzymatic and fluorescence activity based techniques to assess the impact of PAH mutations on pre-mRNA splicing, nonsense-mediated decay (NMD) and receptor complex interactions. We demonstrate that nonsense and frameshift mutations trigger NMD, providing further evidence that haplo-insufficiency is a major molecular consequence of disease-related BMPR2 mutations. We identified heterogeneous functional defects in BMPR-II activity, including impaired type I receptor phosphorylation, receptor interactions and altered receptor complex stoichiometry leading to perturbation of downstream signalling pathways. Importantly, these studies demonstrate that the intracellular domain of BMPR-II is both necessary and sufficient for receptor complex interaction. Finally and to address the potential for resolution of stoichiometric balance, we investigated an agent that promotes translational readthrough of a BMPR2 nonsense reporter construct without interfering with the NMD pathway. We propose that stoichiometric imbalance, due to either haplo-insufficiency or loss of optimal receptor-receptor interactions impairs BMPR-II mediated signalling in PAH. Taken together, these studies have identified an important target for early therapeutic intervention in familial PAH.