Pervasive transcription is widespread amongst eukaryotic genomes, and produces long noncoding RNAs (lncRNAs) in addition to classically annotated transcripts such as messenger RNAs (mRNAs). LncRNAs are heterogeneous in length and map to intergenic regions or overlap with annotated genes. Analogous to mRNAs, lncRNAs are transcribed by RNA polymerase II, regulated by common transcription factors, and possess 5’ caps and perhaps 3’ poly(A) tails. However, lncRNAs perform distinct functions, acting as scaffolds for ribonucleoprotein complexes or directing proteins to nucleic acid targets. The act of transcribing a lncRNA can also affect the local chromatin environment. Furthermore, whereas mRNAs are predominantly turned over in the cytoplasm, both nuclear and cytoplasmic pathways reportedly participate in lncRNA degradation. In this study, I address the question of when and how lncRNAs and mRNAs are distinguished in the cell. Messenger RNAs interact with a defined series of protein factors governing their production, processing and decay, and I hypothesised that lncRNAs might be similarly regulated. I therefore sought to determine which mRNA-binding proteins, if any, also bind lncRNAs. I reasoned that this would reveal the point at which lncRNAs and mRNAs diverge, and how differences in their biogenesis and turnover equip them for different roles. I selected factors from key stages of mRNA metabolism in Saccharomyces cerevisiae, and identified their transcriptome-wide targets using CRAC (crosslinking and analysis of cDNAs). CRAC can detect interactions with low abundance transcripts under physiological conditions, and reveal where within each transcript a protein is bound. Analyses of binding sites in mature mRNAs and intron-containing pre-mRNAs revealed the order in which the tested factors interact with mRNAs, and which region they bind. The poly(A)-binding protein Nab2 bound throughout mRNAs, consistent with an architectural role, whereas the cytoplasmic decay factors Xrn1 and Ski2 bound to poly(A) tails, which might act as hubs to coordinate turnover. The RNA packaging factors Tho2 and Gbp2, and nuclear surveillance factors Mtr4 and Trf4 bound abundantly to intron-containing premRNAs, indicating that they act during or shortly after transcription. The tested factors bound lncRNAs to various extents. LncRNA binding was most abundant for Mtr4 and Trf4, moderate for Tho2, Gbp2, the cap binding complex component Sto1, and the 3’ end processing factors Nab2, Hrp1 and Pab1, and lowest for Xrn1, Ski2 and the export receptor Mex67. This suggests that early events in lncRNA and mRNA biogenesis are similar, but unlike mRNAs, most lncRNAs are retained and degraded in the nucleus. Analyses of two documented classes of lncRNA, cryptic unstable transcripts (CUTs) and stable unannotated transcripts (SUTs), revealed some differences. SUTs were most similar to mRNAs, with canonical cleavage and polyadenylation signals flanking their 3’ ends, and poly(A) tails bound by the poly(A)-binding protein Pab1. CUTs lacked these characteristics, and in comparison to SUTs bound more abundantly to Mtr4 and Trf4 and less so to Ski2, Xrn1 and Mex67. Furthermore, CUTs accumulated upon Hrp1 depletion, suggesting that Hrp1 functions non-canonically to promote CUT turnover. Mtr4, Trf4 and Nab2 also bound abundantly to promoter-proximal RNA fragments generated from ~1000 protein coding genes. These fragments possessed short oligo(A) tails (hallmarks of nuclear surveillance substrates), were not bound to cytoplasmic factors, and apparently correspond to a population of ~150-200 nt promoter-proximal lncRNAs. Notably, CRAC analyses of Mtr4 and Sto1 targets in yeast subjected to a media shift revealed widespread changes in the abundance and surveillance of mRNAs, promoter-proximal transcripts and CUTs, which at many loci were arranged in a complex transcriptional architecture. Overall, the transcriptome-wide binding analyses presented here reveal that lncRNAs diverge from mRNAs prior to export, and are predominantly retained in the nucleus. Transcript fate is apparently determined during 3’ end processing, with CUTs diverging from mRNAs early in transcription via a distinct termination pathway coupled to rapid turnover, and SUTs diverging during or shortly after cleavage and polyadenylation, making them more stable and perhaps prone to escape to the cytoplasm. Promoter-proximal transcripts might arise from termination associated with an early checkpoint in Pol II transcription. The diverse behaviours of lncRNAs arise from their association with distinct subsets of RNA binding proteins, some of which perform different roles when bound to different types of transcript. In conclusion, my results provide the foundation for a mechanistic understanding of how distinct classes of non-coding Pol II transcripts are produced, and how they can perform diverse functions throughout the nucleus.

Evidências sugerem que somente cerca de 2% do genoma codifica proteínas, mas que a maior parte dos 80% restante possui atividade transcricional. Por não ser codificadora de proteínas, essa fração do genoma foi considerada como \'DNA lixo\'. Entretanto, estudos mais recentes e análises pós-ENCODE vem demonstrando que parte significativa destes RNAs não-codificantes desempenham papéis importantes em processos biológicos essenciais e também em doenças. Os RNAs longos não codificadores (lncRNAs) embora tradicionalmente conhecidos pelo imprintinggenômico, vem demonstrando diversos mecanismos de regulação da expressão gênica, principalmente emnível pós transcricional. Um destes lncRNAs que está envolvido principalmente com a metastase em câncer é o HOTAIR. O melanoma tem sido utilizado como modelo de progressao do câncer por suas etapas bem definidas e por isso já tem apresentado alguns lncRNAs envolvidos na melanomagenese e progressão do melanoma, tal como o HOTAIR. Assim, neste trabalho foi analisado a expressão de lncRNAs de amostras de melanócito e melanoma, sendo que as amostras malignas representam as principais fases de progressão deste tipo de câncer. Foram analisados os níveis de expressão relativa. Além disso, foi realizado a expressão diferencial dos grupos representativos do melanoma. Foram encontrados lncRNAs com valores de expressão e significância (p-ajustado <0,01 e fold change >1) que podem ser indicativos de expressão associada a progressão do melanoma. Os lncRNAs mais diferencialmente expressos foram avaliados quanto a sua capacidade de interação proteína-RNA e literatura científica disponível e então foram selecionados para posteriores ensaios funcionais. / Evidence suggests that only about 2% of the genome encodes protein, but most remaining 80% has transcriptional activity. Since they do not coding for proteins, this fraction of the genome was considered \'junk DNA\', However, recent studies and post-ENCODE analisys has shown that significant part of these non-coding RNAs play important roles in essential biological processes and in disease. Long noncoding RNAs (lncRNAs) although traditionally known for genomic imprinting, has demonstrated several mechanisms of regulation of gene expression, especially at the post transcriptional level. One of these lncRNAs that is involved primarily with metastasis in câncer is HOTAIR. Melanoma has been used as a model of câncer progression by its well-defined steps, and so it has been presented some lncRNAs involved in melanoma progression and melanomagenese, as HOTAIR was demonstrated. In this work it was analyzed the expression of lncRNAs of melanocyte and melanoma samples, and malignant samples represent the main stages of progression of this type of câncer. Relative expression levels were analyzed. Furthermore, it was performed differential expression of representative melanoma groups. lncRNAs found with expression values and significance (p-adjusted <0.01 and fold change> 1) may be indicative of expression associated with melanoma progression. The lncRNAs more differentially expressed were evaluated for their ability to interact protein-RNA and available scientific literature and then were selected for further functional assays.

Evidências sugerem que somente cerca de 2% do genoma codifica proteínas, mas que a maior parte dos 80% restante possui atividade transcricional. Por não ser codificadora de proteínas, essa fração do genoma foi considerada como \'DNA lixo\'. Entretanto, estudos mais recentes e análises pós-ENCODE vem demonstrando que parte significativa destes RNAs não-codificantes desempenham papéis importantes em processos biológicos essenciais e também em doenças. Os RNAs longos não codificadores (lncRNAs) embora tradicionalmente conhecidos pelo imprintinggenômico, vem demonstrando diversos mecanismos de regulação da expressão gênica, principalmente emnível pós transcricional. Um destes lncRNAs que está envolvido principalmente com a metastase em câncer é o HOTAIR. O melanoma tem sido utilizado como modelo de progressao do câncer por suas etapas bem definidas e por isso já tem apresentado alguns lncRNAs envolvidos na melanomagenese e progressão do melanoma, tal como o HOTAIR. Assim, neste trabalho foi analisado a expressão de lncRNAs de amostras de melanócito e melanoma, sendo que as amostras malignas representam as principais fases de progressão deste tipo de câncer. Foram analisados os níveis de expressão relativa. Além disso, foi realizado a expressão diferencial dos grupos representativos do melanoma. Foram encontrados lncRNAs com valores de expressão e significância (p-ajustado <0,01 e fold change >1) que podem ser indicativos de expressão associada a progressão do melanoma. Os lncRNAs mais diferencialmente expressos foram avaliados quanto a sua capacidade de interação proteína-RNA e literatura científica disponível e então foram selecionados para posteriores ensaios funcionais. / Evidence suggests that only about 2% of the genome encodes protein, but most remaining 80% has transcriptional activity. Since they do not coding for proteins, this fraction of the genome was considered \'junk DNA\', However, recent studies and post-ENCODE analisys has shown that significant part of these non-coding RNAs play important roles in essential biological processes and in disease. Long noncoding RNAs (lncRNAs) although traditionally known for genomic imprinting, has demonstrated several mechanisms of regulation of gene expression, especially at the post transcriptional level. One of these lncRNAs that is involved primarily with metastasis in câncer is HOTAIR. Melanoma has been used as a model of câncer progression by its well-defined steps, and so it has been presented some lncRNAs involved in melanoma progression and melanomagenese, as HOTAIR was demonstrated. In this work it was analyzed the expression of lncRNAs of melanocyte and melanoma samples, and malignant samples represent the main stages of progression of this type of câncer. Relative expression levels were analyzed. Furthermore, it was performed differential expression of representative melanoma groups. lncRNAs found with expression values and significance (p-adjusted <0.01 and fold change> 1) may be indicative of expression associated with melanoma progression. The lncRNAs more differentially expressed were evaluated for their ability to interact protein-RNA and available scientific literature and then were selected for further functional assays.

RNAs não codificadores longos (lncRNAs) compõem uma fração significativa do transcriptoma. Alterações na expressão de lncRNAs já foram observadas em vários cânceres humanos, mas ainda não foram exploradas no adenocarcinoma pancreático ductal (PDAC), uma doença devastadora e agressiva para a qual faltam métodos para diagnóstico precoce e tratamentos efetivos. Utilizando uma plataforma de microarranjo de cDNA com sondas para 984 lncRNAs e 2371 mRNAs, o presente estudo identificou conjuntos de lncRNAs expressos em 38 amostras clínicas pancreáticas. O enriquecimento de (i) elementos regulatórios associados às regiões promotoras (H3K4me3); (ii) possíveis inícios de transcrição (CAGE-tags); (iii) presença de elementos conservados sugere que ao menos uma fração desses RNAs seja originada a partir de unidades transcricionais independentes, reguladas e possivelmente funcionais. Foram identificadas assinaturas de expressão gênica compostas por mRNA e lncRNAs associadas ao tumor primário e à metástase pancreática. A assinatura gIenica associada à metástase apresentou enriquecimento RNAs intrônicos de loci gênicos associados à via MAPK quinase. O aumento de expressão dos transcritos intrônicos dos loci PPP3CB, MAP3K14 e DAPK1 foi confirmado por qPCR em metástases. Em conjunto, este trabalho aponta para a importância de lncRNAs intrônicos no PDAC e para a necessidade de estudos mais aprofundados para uma melhor compreensão do papel dessa classe de transcritos na biologia da doença. / Long noncoding RNAs (lncRNAs) compose a significant fraction of transcriptome. Altered expression of lncRNAs has been observed in diverse human cancers, but has not being investigated in pancreatic ductal adenocarcinoma (PDAC), a devastating and aggressive disease that lack early diagnosis methods and effective treatments. Using a cDNA microarray platform with probes interrogating 984 lncRNAs and 2371 mRNA, the present study identified subsets of lncRNAs expressed in 38 pancreatic clinical samples. Enrichment of (i) regulatory elements associated to promoter region (H3K4me3); (ii) putative transcription start site (CAGEtags) and (iii) conserved elements, suggest that at least a fraction of these RNAs could be independent transcriptional unit, regulated, an possibly functional. Gene expression signatures comprised of mRNAs and lncRNAs and associated to primary or metastatic tumors were found. A gene signature associated to metastasis was enriched in intronic ncRNAs mapping to gene loci associated to the MAPK pathway. Over expression of intronic RNAs from PPP3CB, MAP3K14 and DAPK1 was confirmed by qPCR in metastatic samples. Taken together, this study points to the importance of intronic lncRNAs in PDAC and for the need to study this class of ncRNAs in greater detail to better understand its role in the biology of PDAC.

RNAs não codificadores longos (lncRNAs) compõem uma fração significativa do transcriptoma. Alterações na expressão de lncRNAs já foram observadas em vários cânceres humanos, mas ainda não foram exploradas no adenocarcinoma pancreático ductal (PDAC), uma doença devastadora e agressiva para a qual faltam métodos para diagnóstico precoce e tratamentos efetivos. Utilizando uma plataforma de microarranjo de cDNA com sondas para 984 lncRNAs e 2371 mRNAs, o presente estudo identificou conjuntos de lncRNAs expressos em 38 amostras clínicas pancreáticas. O enriquecimento de (i) elementos regulatórios associados às regiões promotoras (H3K4me3); (ii) possíveis inícios de transcrição (CAGE-tags); (iii) presença de elementos conservados sugere que ao menos uma fração desses RNAs seja originada a partir de unidades transcricionais independentes, reguladas e possivelmente funcionais. Foram identificadas assinaturas de expressão gênica compostas por mRNA e lncRNAs associadas ao tumor primário e à metástase pancreática. A assinatura gIenica associada à metástase apresentou enriquecimento RNAs intrônicos de loci gênicos associados à via MAPK quinase. O aumento de expressão dos transcritos intrônicos dos loci PPP3CB, MAP3K14 e DAPK1 foi confirmado por qPCR em metástases. Em conjunto, este trabalho aponta para a importância de lncRNAs intrônicos no PDAC e para a necessidade de estudos mais aprofundados para uma melhor compreensão do papel dessa classe de transcritos na biologia da doença. / Long noncoding RNAs (lncRNAs) compose a significant fraction of transcriptome. Altered expression of lncRNAs has been observed in diverse human cancers, but has not being investigated in pancreatic ductal adenocarcinoma (PDAC), a devastating and aggressive disease that lack early diagnosis methods and effective treatments. Using a cDNA microarray platform with probes interrogating 984 lncRNAs and 2371 mRNA, the present study identified subsets of lncRNAs expressed in 38 pancreatic clinical samples. Enrichment of (i) regulatory elements associated to promoter region (H3K4me3); (ii) putative transcription start site (CAGEtags) and (iii) conserved elements, suggest that at least a fraction of these RNAs could be independent transcriptional unit, regulated, an possibly functional. Gene expression signatures comprised of mRNAs and lncRNAs and associated to primary or metastatic tumors were found. A gene signature associated to metastasis was enriched in intronic ncRNAs mapping to gene loci associated to the MAPK pathway. Over expression of intronic RNAs from PPP3CB, MAP3K14 and DAPK1 was confirmed by qPCR in metastatic samples. Taken together, this study points to the importance of intronic lncRNAs in PDAC and for the need to study this class of ncRNAs in greater detail to better understand its role in the biology of PDAC.