Investigating the role of Neurospora RNA-Dependent RNA Polymerase-3 in quelling and meiotic silencing by unpaired DNA

Abstract

RNA interference (RNAi) is a conserved defense system against viruses and transposons which is mediated by small non-coding RNAs. The filamentous fungus Neurospora crassa has served as a valuable model for RNAi studies that greatly expand our knowledge on this complex phenomenon. In Neurospora, there are two post-transcriptional gene silencing pathways, quelling occurs during vegetative growth and is triggered by the presence of transgenes, whereas meiotic silencing by unpaired DNA (MSUD) occurs during meiosis and is induced by unpaired DNA. RNA-dependent RNA polymerases (RdRP) are key components of RNAi pathways responsible for the synthesis of double-stranded RNA. The Neurospora genome encodes three RdRPs; QDE-1 and SAD-1 are required for quelling and MSUD respectively, while the function of the third RdRP is unknown. It is interesting to ask why Neurospora possesses multiple RdRPs and what role these RdRPs play in gene silencing.In this work, I used classical genetic and molecular biology techniques to establish whether RdRP-3 plays a role in quelling or/and in MSUD. Phylogenetic analysis of RdRP proteins from different organisms places RdRP-3 in a group containing Cyptococcus neoformas RDP1 that is known to be involved in RNAi. However, when only the highly conserved RdRP-domain is used, RdRP-3 is with higher confidence, placed in a group with fungal RdRPs of unknown function. Results of quelling and MSUD assays show that the rdrp-3Delta mutant does not affect silencing efficiency of either quelling or MSUD. Hence, tools that can be used to aid further charaterisation of RdRP-3 were constructed. RdRP-3 was tagged with gfp at its endogenous locus to allow the study of RdRP-3's cellular localisation. This strain exhibited growth defects and unfortunately no GFP signal was detected in hyphae or conidia by confocal microscopy. Additionally, because it has been previously shown that rdrp-3 is a dsRNA-activated gene highly induced in the presence of dsRNA, I generated strains expressing albino-1 dsRNA from an inducible promoter. This construct was placed in the wild-type, rdrp-3Delta mutant and in the rdrp-3gfp-tagged background, and the system was validated by RT-PCR analysis. When dsRNA albino-1 was induced in the rdrp-3gfp-tagged background still no GFP signal was detected. Nevertheless, in the WT and in the rdrp-3Delta, upon induction of albino-1 dsRNA, albino-1 mRNA levels decrease and rdrp-3 and NCU07036 mRNA increase as expected. These strains will be used in future to compare gene expression in response to dsRNA in the presence and absence of rdrp-3. In conclusion, the function of RdRP-3 still largely remains unknown, tools created here can be used to explore the possible function of RdRP-3 in response to dsRNA.

Date of Award	1 Aug 2014
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Susan Crosthwaite (Supervisor) & Matthew Ronshaugen (Supervisor)