2 resultados para Small RNA

em CORA - Cork Open Research Archive - University College Cork - Ireland


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Crohn’s disease (CD) is a chronic, relapsing inflammatory condition affecting the gastrointestinal tract of humans, of which there is currently no cure. The precise etiology of CD is unknown, although it has become widely accepted that it is a multifactorial disease which occurs as a result of an abnormal immune response to commensal enteric bacteria in a genetically susceptible host. Recent studies have shown that a new group of Escherichia coli, called Adherent Invasive Escherichia coli (AIEC) are present in the guts of CD patients at a higher frequency than in healthy subjects, suggesting that they may play a role in the initiation and/or maintenance of the inflammation associated with CD. Two phenotypes define an AIEC and differentiate them from other groups of E. coli. Firstly, AIEC can adhere to and invade epithelial cells; and secondly, they can replicate in macrophages. In this study, we use a strain of AIEC which has been isolated from the colonic mucosa of a CD patient, called HM605, to examine the relationship between AIEC and the macrophage. We show, using a systematic mutational approach, that while the Tricarboxylic acid (TCA) cycle, the glyoxylate pathway, the Entner-Doudoroff (ED) pathway, the Pentose Phosphate (PP) pathway and gluconeogenesis are dispensable for the intramacrophagic growth of HM605, glycolysis is an absolute requirement for the ability of this organism to replicate intracellularly. We also show that HM605 activates the inflammasome, a major driver of inflammation in mammals. Specifically, we show that macrophages infected with HM605 produce significantly higher levels of the pro-inflammatory cytokine IL-1β than macrophages infected with a non-AIEC strain, and we show by immunoblotting that this is due to cleavage of caspase-1. We also show that macrophages infected with HM605 exhibit significantly higher levels of pyroptosis, a form of inflammatory cell death, than macrophages infected with a non-AIEC strain. Therefore, AIEC strains are more pro-inflammatory than non-AIEC strains and this may have important consequences in terms of CD pathology. Moreover, we show that while inflammasome activation by HM605 is independent of intracellular bacterial replication, it is dependent on an active bacterial metabolism. Through the establishment of a genetic screen aimed at identifying mutants which activate the inflammasome to lower levels than the wild-type, we confirm our observation that bacterial metabolism is essential for successful inflammasome activation by HM605 and we also uncover new systems/structures that may be important for inflammasome activation, such as the BasS/BasR two-component system, a type VI secretion system and a K1 capsule. Finally, in this study, we also identify a putative small RNA in AIEC strain LF82, which may be involved in modulating the motility of this strain. Overall this works shows that, in the absence of specialised virulence factors, the ability of AIEC to metabolise within the host cell may be a key determinant of its pathogenesis.

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RNA editing is a biological phenomena that alters nascent RNA transcripts by insertion, deletion and/or substitution of one or a few nucleotides. It is ubiquitous in all kingdoms of life and in viruses. The predominant editing event in organisms with a developed central nervous system is Adenosine to Inosine deamination. Inosine is recognized as Guanosine by the translational machinery and reverse-transcriptase. In primates, RNA editing occurs frequently in transcripts from repetitive regions of the genome. In humans, more than 500,000 editing instances have been identified, by applying computational pipelines on available ESTs and high-throughput sequencing data, and by using chemical methods. However, the functions of only a small number of cases have been studied thoroughly. RNA editing instances have been found to have roles in peptide variants synthesis by non-synonymous codon substitutions, transcript variants by alterations in splicing sites and gene silencing by miRNAs sequence modifications. We established the Database of RNA EDiting (DARNED) to accommo-date the reference genomic coordinates of substitution editing in human, mouse and fly transcripts from published literatures, with additional information on edited genomic coordinates collected from various databases e.g. UCSC, NCBI. DARNED contains mostly Adenosine to Inosine editing and allows searches based on genomic region, gene ID, and user provided sequence. The Database is accessible at http://darned.ucc.ie RNA editing instances in coding region are likely to result in recoding in protein synthesis. This encouraged me to focus my research on the occurrences of RNA editing specific CDS and non-Alu exonic regions. By applying various filters on discrepancies between available ESTs and their corresponding reference genomic sequences, putative RNA editing candidates were identified. High-throughput sequencing was used to validate these candidates. All predicted coordinates appeared to be either SNPs or unedited.