Background: The glycolytic pathway is the primary metabolic process which is essential to metabolize a wide range of carbon sources in fungi. Hence, it is important for fungal survival to optimize the level of glycolytic genes expression in response to various environments. Interestingly, in Aspergillus oryzae, several genes which are involved in both glycolysis and gluconeogenesis, have alternative transcription start sites (TSSs). In addition, we demonstrated that selection of TSSs was dependent on two types of carbon sources; one is metabolized via glycolysis such as glucose and fructose, and another via gluconeogenesis such as acetate and ethanol. Particularly, stringent selection of alternative TSS was observed in the enolase-encoding gene (enoA). The similar transcriptional control of enolase gene was suggested in Aspergillus nidulans (Hynes et al., 2007). These findings seem to be important because it provides novel insight into environmental adaptation in the stage of transcriptional regulation of glycolytic genes in Aspergillus spp. However, the molecular details of alternative TSS selection remain to be elucidated. Remarkably, in enoA, there is an intron containing a downstream TSS (dTSS) within 5′ untranslated region (5′ UTR) when it is transcribed from an upstream TSS (uTSS). The length of the intron is 440 bp and quite long in fungi. In this study, we investigated the importance of this 5′ UTR intron in enoA expression.
Results: To this end, we analyzed the enoA promoter plus 5′ UTR whose intron was deleted (PenoA-Δi) or mutated at a splice site (PenoA-issm) using GUS reporter system. Under culture condition with acetate where transcription from uTSS is induced while transcription from dTSS is suppressed, both the GUS activity and the mRNA level were significantly decreased in PenoA-Δi. On the other hand, in PenoA-issm, the mRNA level was unaffected but the GUS activity was almost lost, presumably caused by emergence of uORF within intron unspliced. These results indicated that the deletion of dTSS within intron does not contribute to the reduction of gene expression in PenoA-Δi. Additionally, when the enoA gene was expressed by PenoA-Δi under condition that a resident enoA expression is suppressed, a resulting strain showed a significant reduction of the mRNA transcribed from uTSS and a poor growth under acetate culture condition. These results suggested that 5′ UTR intron enhances the enoA transcription from uTSS in A. oryzae.
Full conference title:
- Asperfest 14 (2017)