Aspergillus oryzae has been paid an attention as a favorable host organism for heterologous protein production. However, despite using a strong expression promoter, heterologous gene expression sometimes results in low mRNA level. We showed previously, using a mite allergen protein, Der f 7, as a model, that premature polyadenylation within ORF of the heterologous gene occurred but was prevented by codon optimization in A.oryzae. In addition, insertion of an internal stop codon into the glucoamylase-Der f 7 (native) fusion gene resulted in the increase in the amount of the transcript. These observations suggested that low mRNA level of the heterologous gene is caused by the degradation process of mRNA lacking translation termination codon, which is called ’nonstop mRNA decay’ pathway. In this study, we analyze the mechanism of nonstop mRNA decay pathway in A.oryzae. Nonstop mRNA decay mechanism has been studied mainly in Saccharomyces cerevisiae, and Ski7p-exosome-dependent 3’í 5’ degradation and Ski7p-independent 5’í 3’ degradation models have been proposed. To elucidate the nonstop mRNA decay mechanism in A. oryzae, we searched for yeast Ski protein homologs in the published Aspergillus genome databases. Interestingly, there are no homologues of Ski7p that is known to be most important for nonstop mRNA decay in yeast, suggesting that nonstop mRNA decay mechanism in filamentous fungi differs from that in yeast. On the other hand, except for Ski7p there are other corresponding Ski protein homologs in Aspergillus genomes, especially Ski1p (5’í 3’ exoribonuclease involved in mRNA decay), Ski2p (one of the components of Ski complex required for 3’í 5’ decay by exosome), and Ski4p (subunit of the exosome) homologs share high identity with those of yeast. Gene disruptants of ski1, ski2, and ski4 were constructed in A. oryzae to examine their involvement in nonstop mRNA decay. Whereas the expression level of nonstop mRNA significantly reduced compared with that of normal mRNA in wild-type and 8710;ski1, this reduction was depressed clearly in 8710;ski4 and also slightly in 8710;ski2. This suggested that nonstop mRNA is degraded by 3’í 5’ decay pathway in A. oryzae.
Full conference title:
9th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 9th (2008)