Resumo

The filamentous fungus Neurospora crassa is an excellent model system for examining molecular responses to ambient signals in eukaryotic microorganisms. Inorganic phosphate (Pi) is an essential growth-limiting nutrient in nature and is crucial for the synthesis of nucleic acids and the flow of genetic information. The genetic and molecular mechanisms controlling the response to Pi starvation in N. crassa include at least four genes (nuc-2, preg, pogv, and nuc-1), which are involved in a hierarchical regulatory activation network. Pi availability is sensed by the nuc-2 gene, the transcription of which is regulated in response to extracellular Pi changes. NUC-2, an ankyrin repeat protein, transmits a metabolic signal downstream of the hierarchical regulatory pathway, inhibiting PREG-PGOV complex function under conditions of Pi shortage. This allows translocation of the transcriptional regulator NUC-1 into the nucleus. PREG is a cyclin-like protein kinase and PGOV is a cyclin-dependent protein kinase, whereas NUC-1 is a member of the basic helix–loop–helix (bHLH) family of transcription factors. Under conditions of Pi shortage, this hierarchical regulatory network removes the repression of genes coding for nucleases, phosphatases, and Pi transporters that are necessary for fulfilling the Pi requirements in the cell. Numerous genes modulated by NUC-2 protein under Pi shortage was previously identified, including the mak-2 gene, suggesting that the MAPK cascade is somehow involved in the phosphate signaling pathway. Moreover, the identification of genes modulated by the nuc-2, preg, and nuc-1 genes revealed various aspects of the phosphorous-sensing network, including molecular events implicated in the preg convergent regulation of Pi, nucleotide, and energy metabolism. In a previous work we performed microarray experiments using a mak-2 knockout strain (Δmak-2) grown under phosphate-shortage conditions (10 µM Pi, pH 5.4, 5 hours cultivation, 30oC) by comparing its transcription profile to that of a control strain grown in low- and high-phosphate cultures. These experiments revealed numerous unique differentially expressed genes involved in a number of physiological processes related to phosphate transport, metabolism, and regulation as well as posttranslational modification of proteins, and MAPK signaling pathways. In this study we show that pre-mRNA splicing of the genes coding for an asparagine sintetase and a farnesil transferase is modulated by the MAPK MAK-2 in response to extracellular phosphate changes. Interestingly, the pre-mRNA transcription processing follows apparently different metabolic routes for these two genes, emphasizing the complexity of the metabolic modulation exerted by the MAPK MAK-2 in N. crassa.