Elucidation of the role of a cryptic RNAi machinery in the human pathogenic yeast candida albicans

Abstract

RNA interference (RNAi) is the phenomenon of degradation of target RNA, mediated by small regulatory RNAs, that results in silencing of chromatin and protection of the cell against invading viruses and endogenous mobile genetic elements. Though the phenomenon was first described in the nematode worm Caenorhabditis elegans by Fire and colleagues, the protein machinery required for RNAi is now known to be present across the prokaryotic and eukaryotic lineages with some species having the full complement of the machinery, others possessing only a subset thereof and some even lacking the machinery altogether. In the taxa in which the machinery is found, one or more pathways of RNAi may be functional. These pathways are distinguished based on the small regulatory RNA involved, and are named as the microRNA, small interfering RNA and PIWI-interacting RNA pathways based on the small RNA they utilize. These pathways carry out one or more of the functions associated with the RNAi machinery. Barring a few exceptions, the protein machinery required for RNAi is conserved across the three pathways. Of the entire complement of RNAi proteins, two are unambiguously recognized to be present at the heart of silencing process. These two proteins, Dicer and Argonaute, are required for RNAi in most eukaryotic organisms. Dicer performs the function of generation of the small regulatory RNAs while Argonaute is involved in target recognition using these small RNAs and in downstream effector functions. Originally known for its role in chromatin silencing and cellular defence, novel functions of the RNAi machinery in genome maintenance, transcription and processing of RNA species have been discovered, highlighting its functional diversity. The fungi are a major lineage of the Eukarya with enormous diversity in ecology, morphology and lifecycles. As is the case with other eukaryotes, the RNAi machinery found in various fungal taxa is also functionally diverse. The non-canonical functions include quelling, DNA repair and meiotic silencing of unpaired DNA in Neurospora crassa, pathogenesis in Botrytis cinerea and sex-induced silencing in Cryptococcus neoformans to name a few. The diversity in RNAi in the fungal kingdom is not restricted to the functions of the machinery alone but also extends to the domain architecture of the proteins involved. This is exemplified by the non-canonical Dicer of the budding yeasts. This protein has retained only the catalytic RNase III domain and the double stranded RNA binding domain of its higher eukaryotic counterparts, yet is able to perform the same function of small RNA generation as them, albeit with a slightly different mechanism. Budding yeasts also possess an Argonaute protein, which along with Dicer comprises the RNAi machinery of these yeasts. The budding yeast Argonaute resembles the Argonautes of the higher eukaryotes in domain architecture and presumably functions by the same mechanism. Candida albicans is the most commonly isolated yeast pathogen of humans. This budding yeast, like some others in its group, possesses the non-canonical Dicer and Argonaute proteins. Even though, endogenous silencing of genes by these proteins has been examined, the results of these studies are inconclusive. Given the diverse array of functions RNAi is known to perform in fungi, it is conceivable that the RNAi machinery of C. albicans may be performing additional roles aside from that in gene silencing. An earlier study has explored other possible functions associated with this machinery, but not extensively. The aim of this project is to assess the capability of the RNAi machinery of C. albicans to silence genes and to investigate other functions, if any, of the machinery in this organism. Specifically, we were interested to determine whether the machinery is involved in controlling position-effect variegation at the C. albicans centromeres, drawing parallels from the closely related fission yeast Schizosaccharomyces pombe. This thesis deals with results pertaining functions of the RNAi machinery in C. albicans other than that in gene silencing. During the course of this study, it was discovered that absence of Dicer results in growth retardation of C. albicans which was further observed to be function of temperature. This slow growth was determined to arise not due to cell mortality but instead probably due to an increase in the time taken to complete the cell cycle itself. Such a link between cell cycle and RNase III enzymes is not unique and has been shown in other species as well. From analysis of flow cytometry plots it was proposed that this delay is presumably occurring in the S phase of the cycle as the plots of the mutant showed an accumulation of cells in the S-phase as compared to the wild type. To test if the processes of DNA replication or DNA repair, the major processes in the S-phase, were affected in the absence of Dicer, the mutant cells were treated with the replication inhibitor hydroxyurea (HU) and oxidizing agent dimethyl sulfoxide (DMSO). The mutants were found to be a fold more sensitive to DMSO indicating that Dicer has a role to play in protecting the cell against oxidative damage. This role however, cannot yet be associated with repair of damaged DNA as oxidative damage by DMSO is not specific to DNA. It is however possible that such a scenario may exist as Dicer is known to be involved in DNA repair in other species. Upon treatment with HU, the mutants were found to be resistant to the drug as compared to the wild type. As of now, we are unsure of what is the cause for such a phenotype and can only list few possibilities which could result in HU resistance. Absence of Dicer was also observed to lead to an increase in cellular size. Deletion of Argonaute did not affect growth as was observed with Dicer. We also did not see any of the other phenotypes upon Argonaute deletion that were observed for Dicer depletion. Further, there is no other protein with domain architecture similar to Argonaute present in the C. albicans genome which could carry out Argonaute’s function in its absence thus ruling out the possibility of redundancy. Based on these facts, we believe that in C. albicans Dicer and Argonaute may have functionally diverged with respect to the above mentioned phenotypes. In S. pombe, position-effect variegation is exerted on transgenes inserted in the centromeres. Deletion of RNAi genes of S. pombe alleviates this position-effect variegation, thus implicating RNAi in controlling the phenomenon. Position-effect variegation is also seen on transgenes inserted in C. albicans centromeres. We wanted to ascertain whether, like in the closely related ascomycete S. pombe, RNAi also affects this phenomenon at the C. albicans centromeres. We have found that in the absence of either Dicer or Argonaute, this phenomenon is unaffected at C. albicans centromeres, thus ruling out a function similar to that observed in S. pombe. In conclusion, in this work we have attempted to characterize the functions of the RNA interference proteins of the human fungal pathogen C. albicans. We have observed some phenotypes associated with absence of Dicer and based on them speculate that it may be playing a role in the cell cycle. Deletion of Argonaute does not confer the same phenotypes as depletion of Dicer; this fact along with other evidences suggests that they may have functionally diverged. Further, we have found that unlike in S. pombe, the position-effect variegation observed at the C. albicans centromeres is not under the control of the RNAi machinery.