Identificación y estudio funcional de una proteína de choque térmico implicada en el mecanismo no canónico de silenciamiento génico de Mucor lusitanicus

Abstract

Gene silencing was discovered in 1990 in plants. Later, it was discovered in animal and fungi. Since then, gene silencing has been the focus of many scientific studies and several pathways observed in nature have prompted a great interest. Double-stranded RNA (dsRNA) molecules provoke the activation of the silencing mechanism or RNAi (RNA interference), whose origin may be endogenous or exogenous. This mechanism allows the recognition of the complementary strand of the guide RNA, which are generated from dsRNA. Thus, the silencing mechanism can cut the target RNA. There are two different pathways in Mucor lusitanicus, a canonical pathway and a non-canonical one, named NCRIP. The ribonuclease R3B2, which is a very unusual protein of Mucorales that only cuts single-stranded RNA, has a main role in NCRIP. In this work, the interactome of R3B2 was identified by means of a yeast two-hybrid screening using a cDNA library of M. lusitanicus. In this assay, a heat shock protein belonging to the Hsp70 family was identified. This protein is a member of the HspA12 subfamily, the most atypical group of Hsp70. The HspA12 group shows strong differences in the domain architecture comparing with the rest of the subfamilies. Diverse phylogenetic analyses showed that this group of proteins is conserved and has undergone an expansion in some species, as M. lusitanicus. A complementary yeast two-hybrid assay identified the regions of R3B2 and HspA12a involved in the interaction and showed that none of the rest of proteins that participates in the silencing mechanism interact with HspA12a. Furthermore, using the protoplast transformation method, two mutants in hspA12a were generated by homologous recombination. These two independent mutants grew poorly at 30 ºC, but correctly at 26 ºC, the optimal temperature for M. lusitanicus. Some colonies that could grow at 30 ºC (less than 1 % of the spores) were able to transmit this survival capacities to its spores, producing a higher percentage of colonies at 30 ºC in the next cycles (30 %). This phenotype disappeared after some vegetative cycles at 26 ºC, which could mean that this phenomenon is regulated by epigenetic mechanisms. During these assays, some individuals were able to grow perfectly at 30 ºC. Their genomes were sequenced and analyzed, revealing the presence of several mutations that were absent in the parental strain genome. Some genes were identified as candidate genes for that phenomenon. The analysis of hspA12a expression showed that it is increased when the temperature was higher (30 ºC). Moreover, the expression of two different reporter genes of NCRIP in the mutants proved that hspA12a is essential for the pathway at 30 ºC, but not at 26 ºC. In addition, its participation in the canonical pathway and the implication of the region involved in the interaction with R3B2 in the ribonuclease activity were studied, without positive results. Finally, a virulence assay with mice showed that the hspA12a mutants were less virulent that the virulent control, both mutants showing results closer to the avirulent strain. In conclusion, it has been discovered a new protein of the silencing mechanism of M. lusitanicus. This protein participates in NCRIP (probably stabilizing R3B2 under stress conditions), is linked to the resistance at high temperatures, and may be involved in the pathogenicity of this fungus.