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.