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Title Identifying components of the non-Canonical RNA silencing mechanism in Mucor circinelloides = Identificación de componentes del mecanismo no canónico de silenciamiento mediado por RNA en Mucor circinelloides
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Date Accessioned
Discipline/Department Departamento de Genética y Microbiología
University/Publisher Universidad de Murcia
Abstract The increasing knowledge on the functional relevance of endogenous small RNAs (esRNAs) as riboregulators has stimulated the identification and characterization of these molecules in numerous eukaryotes. In the basal fungus Mucor circinelloides, an emerging opportunistic human pathogen, esRNAs that regulate the expression of many protein coding genes have been described. These esRNAs share common machinery for their biogenesis consisting of an RNase III endonuclease Dicer, a single Argonaute protein and two RNA-dependent RNA polymerases. Besides participating in this canonical dicer-dependent RNA interference (RNAi) pathway, the M. circinelloides rdrp genes are involved in the production of a novel dicer-independent esRNA class, which showed a very strong strand bias, being exclusively sense to the mRNAs, and a random spread of size distribution, suggesting that they are degradation products of endogenous mRNAs. The overall objective of this thesis is to characterize this putative mRNA degradation pathway, identify the RNase involved and use the Mucor silencing mechanisms for whole-genome functional analysis. These global objectives are specified in the following: Objectives: i) Functional analysis of the rdrp-dependent dicer-independent pathway in the regulation of gene expression. ii) In silico identification of M. circinelloides candidate RNases to be involved in the dicer-independent degradation pathway. iii) Functional studies of the candidate genes in the production of rdrp-dependent dicer-independent esRNAs. iv) Construction of genomic libraries for functional analysis by knocking-down genes using silencing vectors to identify M. circinelloides sequences with putative roles in pathogenesis. v) Generation of null mutants for each candidate gene to confirm the phenotype and investigate their roles in Mucor pathogenesis. Methods: in vivo and in vitro genetic manipulations were used to analyze gene functions. These include methods to isolate, amplify and analyze the expression of specific genes and genetic transformation of living cells. Genomic DNA libraries were constructed in silencing vectors containing dual promoters for whole-genome functional analysis and identification of candidate genes with a possible role in Mucor pathogenesis. Phenotypic analyses were used to evaluate the functions of candidate genes in growth, morphogenesis and virulence of this fungus. Results: Expression analysis demonstrated that the new rdrp-dependent dicer-independent pathway regulates gene expression by promoting the specific degradation of mRNAs by a previously unknown RNase. This pathway mainly regulates conserved genes involved in metabolism and cellular processes and signaling, such as those required for heme biosynthesis, and controls responses to specific environmental signals. Searching the Mucor genome for candidate RNases to participate in this pathway, and functional analysis of the corresponding knockout mutants identified a new protein, R3B2, which is only found in basal fungi. This RNase III-like protein participates…
Subjects/Keywords Hongos-Genética; Ciencias; 58
Contributors [email protected] (authoremail); false (authoremailshow); Ruiz Vázquez, Rosa Mª (director); Nicolás Molina, Francisco Esteban (director); true (authorsendemail)
Language en
Rights ADVERTENCIA. El acceso a los contenidos de esta tesis doctoral y su utilización debe respetar los derechos de la persona autora. Puede ser utilizada para consulta o estudio personal, así como en actividades o materiales de investigación y docencia en los términos establecidos en el art. 32 del Texto Refundido de la Ley de Propiedad Intelectual (RDL 1/1996). Para otros usos se requiere la autorización previa y expresa de la persona autora. En cualquier caso, en la utilización de sus contenidos se deberá indicar de forma clara el nombre y apellidos de la persona autora y el título de la tesis doctoral. No se autoriza su reproducción u otras formas de explotación efectuadas con fines lucrativos ni su comunicación pública desde un sitio ajeno al servicio TDR. Tampoco se autoriza la presentación de su contenido en una ventana o marco ajeno a TDR (framing). Esta reserva de derechos afecta tanto al contenido de la tesis como a sus resúmenes e índices.
info:eu-repo/semantics/openAccess
Country of Publication es
Record ID handle:10803/308346
Repository barcelona
Date Indexed 2018-03-21
Issued Date 2015-07-16 00:00:00

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