Orientador: Renato Vicentini dos Santos / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T20:26:13Z (GMT). No. of bitstreams: 1 Canesin_LucasEduardoCosta_M.pdf: 8460459 bytes, checksum: 3fde4864bd253faed92b7dfd9071be3c (MD5) Previous issue date: 2014 / Resumo: Recentemente, a análise de dados de genômica comparativa, buscando elucidar melhor a hipótese nula de modelos evolutivos, i.e. evolução neutra, originou uma nova teoria que eleva o tamanho populacional como principal fator evolutivo. Populações pequenas estão sujeitas a forte influência de deriva genética, o que causa o aumento da entropia do genoma. A complexidade genômica, leia-se conteúdo de sequencias informativas, como genes, é então um subproduto do aumento da entropia e a seleção teria então um papel secundário, sobretudo como moduladora do processo evolutivo. Assumindo este modelo, a emergência e degeneração de transcritos linhagem-específicos estão submetidas primariamente a evolução neutra. A transcrição pervasiva, sobretudo em linhagens germinais, é o agente causal do nascimento de genes e a fixação destes, frente ao reduzido tamanho populacional de eucariotos multicelulares, como as plantas Saccarum officinarum e Sorghum bicolor, ocorre por deriva genética. A inserção de novos genes, que são inicialmente neutros ou levemente deletérios, em redes funcionais ainda é pouco compreendida. A integração se torna gradativamente mais robusta com a evolução individual destes loci. Neste contexto, este estudo buscou identificar genes codificadores e não-codificadores de proteínas de recente emergência em cana-de-açúcar e sorgo a fim de se elucidar a hipótese de que sua arquitetura gênica e integração em redes biológicas apresentam padrões evolutivos comuns. Para isso, realizamos a identificação de lncRNAs de cana a partir de bancos de cDNA, o que permitiu a caracterização da expressão desses transcritos contrastando seis variedades distintas. Em decorrência da disponibilidade do genoma de sorgo, a identificação de genes linhagem-específicos codificadores e não codificadores pode ser resolvida com maior precisão. Pudemos determinar uma correlação entre a sua arquitetura gênica e integração nas redes biológicas e sua idade relativa. Apesar da correlação encontrada, o efeito mais forte observado em transcritos não codificadores revelam outros fatores que devem estar influenciando sua evolução. Levantamos a hipótese de que o evento de tradução possa elevar a eficiência da seleção negativa sobre o transcrito emergente, o que resultaria no turnover mais acentuado de lincRNAs e maior conservação de genes linhagem-específicos / Abstract: Recently, comparative genomics studies, aiming to better elucidate the null hypothesis of models of evolution, i. e. the neutral evolution, originate a new theory that elects the population size as the main factor acting in evolution. Small populations are subject to stronger influence of genetic drift, which raises genomic entropy. Genomic complexity, which means the information content in genome, such as genes, is a byproduct of the high entropy levels and selection would then display a secondary role, mainly as a modulator of the evolutionary process. Assuming this model, the emergence and degeneration of lineage-specific transcripts are primarily subject to neutral evolution. The pervasive transcription, especially in germinal cell lines, is the causal agent of birth of genes and their fixation, in face to the reduced population size of multicellular eukaryotes, as Saccarum officinarum and Sorghum bicolor plant species, is ruled by genetic drift. The integration of new genes, initially neutral or weakly deleterious, in functional networks is still poorly understood. The integration becomes more robust with the individual historical evolutionary path of these loci. In this context, this study aimed identify protein coding and noncoding genes of recent emergence in in sugarcane and sorghum to elucidate the hypothesis that the gene architecture and integration in biological networks display common patterns of evolution. We then identified sugarcane lncRNAs from public cDNA databases that allowed us to characterize the expression of these transcripts in six different contrasting varieties of sugarcane. As sorghum bicolor genome is available, the identification of lineage-specific coding and noncoding could be done to a higher resolution. We could then determine a correlation between gene architecture and network integration with its relative age. Despite the correlation observed, a stronger effect seen in noncoding transcripts reveals other factors that may be influencing their evolution. We propose the hypothesis that the translation event may increase negative selection efficiency over the emerging transcript, what would result in the stronger turnover of lincRNAs and higher conservation levels of coding lineage-specific genes / Mestrado / Bioinformatica / Mestre em Genética e Biologia Molecular

Recent genome-wide sequencing projects revealed the pervasive transcription of intergenic long noncoding RNAs (lincRNAs) in eukaryotic genomes (reviewed in Ponting et al. 2009). For the vast majority of lincRNAs, their mechanisms of function remain largely unrecognized. However, the genome-wide signatures of functionality associated with many lincRNAs, including apparent evolutionary sequence conservation, spatial and temporal-restricted expression patterns, strong associations with epigenetic marks, and reported molecular and cellular functions, reinforce their biological relevance. My work investigates lincRNAs that post-transcriptionally regulate gene abundance by competing for the binding of common microRNAs (miRNAs) with protein-coding transcripts, termed competitive endogenous RNAs (ceRNAs) acting lincRNAs (lnceRNAs). First, I examine the biological relevance of this post-transcriptional regulation of gene abundance by ceRNAs. Next, I estimate the genome-wide prevalence of lnceRNAs in mouse embryonic stem cells (mESCs) and characterize their properties. Finally, using two specific examples of lnceRNAs, I show the contributions of lnceRNAs to human monogenic and complex trait diseases. Collectively, these results illustrate that lnceRNAs provide a novel layer of post-transcriptional regulation via a miRNA-mediated mechanism that contributes to organismal and cellular biology.

The innate immune system is a multicomponent response governed by intricate mechanisms of induction, regulation and resolution to elicit antimicrobial defenses. In recent years, the complexity of eukaryotic transcriptomes has become the subject of intense scrutiny and curiosity. It has been established, that RNA polymerase II (RNAPII) transcribes hundreds to thousands of long noncoding RNAs (lncRNAs), often in a stimulus and cell-type specific manner. However, the functional significance of these transcripts has been particularly controversial. While the number of identified lncRNAs is growing, our understanding of how lncRNAs themselves regulate other genes is quite limited. In chapter 2, a novel lncRNA is identified, more specifically, a natural antisense transcript, that mediates the transcription of the pro-inflammatory cytokine IL-1α. Through loss-of-function studies, I report the necessity of this transcript in mediating IL-1α mRNA expression by affecting RNAPII binding to the IL-1α promoter after toll-like receptor signaling. For the first time, I show that IL-1α is regulated at the transcriptional level. As a second independent component of this thesis, we explore the role of the innate immune response after infection by the malaria-causing parasite, Plasmodium berghei ANKA (PbA), and how innate immune components are both beneficial and detrimental to the host depending on when and where inflammation is triggered during infection. We attempt to identify the “malarial toxin” responsible for aberrations in the immune response that is detrimental for disease outcomes and the innate signaling pathways that are involved. Many pathogens induce pathological inflammatory conditions that lead to irreparable homeostatic imbalances and become fatal to the host. Here, type I Interferon signaling is required to dampen parasite load during liver-stage infections, but leads to host mobidity if these pathways are activated in the erythrocytic phase of infection. Together, this thesis provides new insights on how components of the innate immune system are regulated, and how dysregulation of immunity can potentially lead to adverse effects during active infections.

Eukaryotic genomes can produce two types of transcripts: protein-coding and non-coding RNAs (ncRNAs). Cryptic ncRNA transcripts are bona fide RNA Pol II products that originate from bidirectional promoters, yet they are degraded by the RNA exosome. Such pervasive transcription is prevalent across eukaryotes, yet its regulation and function is poorly understood. We hypothesized that chromatin architecture at cryptic promoters may regulate ncRNA transcription. Nucleosomes that flank promoters are highly enriched in two histone marks: H3-K56Ac and the variant H2A.Z, which make nucleosomes highly dynamic. These histone modifications are present at a majority of promoters and their stereotypic pattern is conserved from yeast to mammals, suggesting their evolutionary importance. Although required for inducing a handful of genes, their contribution to steady-state transcription has remained elusive. In this work, we set out to understand if dynamic nucleosomes regulate cryptic transcription and how this is coordinated with the RNA exosome. Remarkably, we find that H3-K56Ac promotes RNA polymerase II occupancy at a large number of protein coding and noncoding loci, yet neither histone mark has a significant impact on steady state mRNA levels in budding yeast. Instead, broad effects of H3-K56Ac or H2A.Z on levels of both coding and ncRNAs are only revealed in the absence of the nuclear RNA exosome. We show that H2A.Z functions with H3-K56Ac in chromosome folding, facilitating formation of Chromosomal Interaction Domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA levels, perhaps in part by regulating higher order chromatin structures. Together, these chromatin factors achieve a balance of RNA exosome activity (yin; negative) and Pol II (yang; positive) to maintain transcriptional homeostasis.

XIST RNA established the precedent for a noncoding RNA that stably associates with and regulates chromatin, however it remains poorly understood how such RNAs structurally associate with the interphase chromosome territory. I demonstrate that transgenic XIST RNA localizes in cis to an autosome as it does to the inactive X chromosome, hence the RNA recognizes a structure common to all chromosomes. I reassess the prevalent thinking in the field that a single protein, Scaffold Attachment Factor-A (SAF-A/hnRNP U), provides a single molecule bridge required to directly tether the RNA to DNA. In an extensive series of experiments in multiple cell types, I examine the effects of SAF-A depletion or different SAF-A mutations on XIST RNA localization, and I force XIST RNA retention at mitosis to examine the effect on SAF-A. I find that SAF-A is not required to localize XIST RNA but is one of multiple proteins involved, some of which frequently become lost or compromised in cancer. I additionally examine SAF-A’s potential role localizing repeat-rich CoT-1 RNA, a class of abundant RNAs that we show tightly and stably localize to euchromatic interphase chromosome territories, but release upon disruption of the nuclear scaffold. Overall, findings suggest that instead of “tethering” chromosomal RNAs to the scaffold, SAF-A is one component of a multi-component matrix/scaffold supporting interphase nuclear architecture. Results indicate that Cot-1 and XIST RNAs form integral components of this scaffold and are required to maintain the chromosomal association of SAF-A, substantially advancing understanding of how chromatin-associated RNAs contribute to nuclear structure.