The Role of Decapping Factors During Nonsense-Mediated Decay (NMD) in Aspergillus nidulans

RNA degradation is ubiquitous and it is clear that it must be carefully controlled to accurately recognise and target appropriate transcripts. There are several pathways for mRNA degradation and decapping is one of the critical steps in determining transcript stability. The focus of this study was the identification and characterisation of factors involved in decapping and their involvement in nonsense-mediated mRNA decay (NMD) in Aspergillus nidulans. Our studies have shown that disruption of two decapping factors, Dcp1 and the Nudix protein Dcp2, lead only to partial suppression of NMD. This distinguishes A. nidulans from Saccharomyces cerevisiae, where the two decapping factors are required for NMD. Deletion of lsm1, which encodes a component of a heptomeric complex, Lsm1-7, a known promoter of decapping, also partially supressed NMD. To our knowledge this is the first time that the role of Lsm’s in NMD has been described. A similar result was observed when another Nudix family protein, NdxD, was disrupted. We propose that NdxD is a second decapping factor in A. nidulans. Disruption of other factors known to promote decapping and subsequent RNA degradation, Pat1, Dhh1 and Xrn1, did not affect NMD, demonstrating these factors are not required for NMD in A. nidulans. In order to quantify decapping, we set out to establish a simple and reliable assay to quantify the decapped transcripts. The method utilised splinted-ligation through which an RNA adaptor is ligated specifically to the 5’ end of decapped transcripts with the help of splint primer. The primer has a complementary sequences to the RNA adaptor at its 3’ end and the eight random nucleotides at the 5’ end to facilitate hybridisation to any decapped transcript. qRT-PCR was utilised to amplify the ligation products for a specific transcript and used internal primers as a control to assess the relative level of decapped transcripts. This gave a good basis for quantifying the decapped transcripts, however further optimisation is required in order to develop a robust assay. Although it has been known that both Dcp1 and Dcp2 form a decapping complex in yeast, our studies showed that Dcp2 has a significant role in stabilising the uaZ+ transcript, while deletion of dcp1 did not. Fluorescence microscopy has shown that both of these proteins localise primarily to the expected P-body like structures, however, the major proportions of Dcp2 and Dcp1 did not co-localise and were therefore not interacting. These data suggest that the Dcp2 activity is not solely dependent on Dcp1, suggesting a divergence between A. nidulans and S. cerevisiae. Additionally, confocal microscopy was used to characterise the intracellular distribution of CutA and CutB, which are involved in 3’ pyrimidine-tagging of transcripts, promoting decapping and degradation of mRNA. Using GFP and RFP tagged proteins, we determined that CutA is primarily localised in the cytoplasm whereas CutB is primarily, but not exclusively, located in the nuclei. Interestingly deletion of cutB lead to increased levels of CutA in the nuclei suggesting an interplay between the two proteins. Deletion of dcp1 produces an aberrant polysome profile as determined by sucrose gradient centrifugation. The predominant peak correlated with the large (60S) subunit rather than the monosome (80S) peak observed for WT. The small (40S) subunit was also relatively high. These observations distinguished ∆dcp1 from WT and the phenotype of the ∆dcp2 strain was intermediate between the two. The accumulation of 60S peak in ∆dcp1 included a relatively high proportion of 28S rRNA derived fragments. Northern analysis of these putative 60S degradation products and sequencing of two specific fragments suggest that in the ∆dcp1 strain the ribosomes are being cleaved, possibly as part of an rRNA turnover mechanism. Although genetic analysis showed that both ∆dcp2 and a point mutation, dcp2 E148Q, which is likely to disrupt the nuclease activity, are both epistatic to dcp1 with respect to this phenotype. Northern analysis indicates that the degradation products observed in ∆dcp1, ∆dcp2 and WT strains appear very similar, even though the levels vary dramatically. This implies that Dcp2 is probably not directly responsible for these cleavage events or it is one of a number of activities cleaving the rRNA in what appears to be a similar way.