The Stress Response and Circadian Regulation of Translation in Neurospora crassa

Stress response pathways function to allow cells to adapt to changes in the environment. In Neurospora crassa, acute osmotic stress activates the conserved p38-like osmosensing mitogen-activated protein kinase (OS MAPK) pathway. When activated, the terminal MAPK, OS-2 can activate transcription factors and kinases. We show an acute osmotic stress activates OS-2, which phosphorylates and activates the conserved kinase RCK-2. RCK-2 phosphorylates and inactivates the highly conserved eukaryotic elongation factor 2 (eEF-2). To determine if this is a mechanism for translational regulation of mRNAs, I examined ribosome profiling and RNAseq data from osmotically stressed WT and Δrck-2 cultures. I found that RCK-2/eEF-2 regulate 69 constitutively expressed mRNAs at the level of translation. I also examined ribosome profiling and RNAseq data from cultures given light exposure, and found that 36 constitutively expressed mRNAs were regulated at the level of translation. In both cases, the translationally-controlled genes were enriched for metabolic processes, suggesting that rapid regulation of metabolism through translational control helps the organism overcome osmotic and light stress. The circadian clock has a profound effect on gene regulation; however, little is known about the role of the clock in controlling translation. I show that clock signaling through the OS MAPK pathway promotes rhythmic phosphorylation of RCK-2 and eEF-2 in constant conditions. Using a cell-free translation assay, I demonstrated that clock signaling to eEF-2 leads to rhythmic control of mRNA translation. To determine the extent of clock regulation of translation in vivo, I examined ribosome profiling and RNAseq data from WT cultures over a circadian time course. 637 Neurospora mRNAs showed rhythmic ribosome occupancy, and 549 of these were from constitutively expressed mRNAs. To determine which of these translationally cycling mRNAs required RCK-2/eEF-2 regulation, I examined ribosome profiling and RNAseq data from Δrck-2 cultures over a circadian time course. I found 419 of the constitutive mRNAs with cycling ribosomal occupancy required clock regulation of RCK-2/eEF-2. While the regulation of initiation was thought to be the main control point of translation, these data revealed a major role for eEF-2 activity and elongation in translation control following stress and by the circadian clock

Translation initiation from conserved non-AUG codons provides additional layers of regulation and coding capacity

Neurospora crassa cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying transcription activators that drive the transcriptional response to amino acid limitation. The cpc-1 mRNA contains two upstream open reading frames (uORFs) in its >700-nucleotide (nt) 5' leader, and its expression is controlled at the level of translation in response to amino acid starvation. We used N. crassa cell extracts and obtained data indicating that cpc-1 uORF1 and uORF2 are functionally analogous to GCN4 uORF1 and uORF4, respectively, in controlling translation. We also found that the 5' region upstream of the main coding sequence of the cpc-1 mRNA extends for more than 700 nucleotides without any in-frame stop codon. For 100 cpc-1 homologs from Pezizomycotina and from selected Basidiomycota, 5' conserved extensions of the CPC1 reading frame are also observed. Multiple non-AUG near-cognate codons (NCCs) in the CPC1 reading frame upstream of uORF2, some deeply conserved, could potentially initiate translation. At least four NCCs initiated translation in vitro. In vivo data were consistent with initiation at NCCs to produce N-terminally extended N. crassa CPC1 isoforms. The pivotal role played by CPC1, combined with its translational regulation by uORFs and NCC utilization, underscores the emerging significance of noncanonical initiation events in controlling gene expression. IMPORTANCE There is a deepening and widening appreciation of the diverse roles of translation in controlling gene expression. A central fungal transcription factor, the best-studied example of which is Saccharomyces cerevisiae GCN4, is crucial for the response to amino acid limitation. Two upstream open reading frames (uORFs) in the GCN4 mRNA are critical for controlling GCN4 synthesis. We observed that two uORFs in the corresponding Neurospora crassa cpc-1 mRNA appear functionally analogous to the GCN4 uORFs. We also discovered that, surprisingly, unlike GCN4, the CPC1 coding sequence extends far upstream from the presumed AUG start codon with no other in-frame AUG codons. Similar extensions were seen in homologs from many filamentous fungi. We observed that multiple non-AUG near-cognate codons (NCCs) in this extended reading frame, some conserved, initiated translation to produce longer forms of CPC1, underscoring the significance of noncanonical initiation in controlling gene expression

Circadian clock regulation of mRNA translation through eukaryotic elongation factor eEF-2

The circadian clock has a profound effect on gene regulation, controlling rhythmic transcript accumulation for up to half of expressed genes in eukaryotes. Evidence also exists for clock control of mRNA translation, but the extent and mechanisms for this regulation are not known. In Neurospora crassa, the circadian clock generates daily rhythms in the activation of conserved mitogen-activated protein kinase (MAPK) pathways when cells are grown in constant conditions, including rhythmic activation of the well-characterized p38 osmosensing (OS) MAPK pathway. Rhythmic phosphorylation of the MAPK OS-2 (P-OS-2) leads to temporal control of downstream targets of OS-2. We show that osmotic stress in N. crassa induced the phosphorylation of a eukaryotic elongation factor-2 (eEF-2) kinase, radiation sensitivity complementing kinase-2 (RCK-2), and that RCK-2 is necessary for high-level phosphorylation of eEF-2, a key regulator of translation elongation. The levels of phosphorylated RCK-2 and phosphorylated eEF-2 cycle in abundance in wild-type cells but not in cells deleted for OS-2 or the core clock component FREQUENCY (FRQ). Translation extracts from cells grown in constant conditions show decreased translational activity in the late subjective morning, coincident with the peak in eEF-2 phosphorylation, and rhythmic translation of glutathione S-transferase (GST-3) from constitutive mRNA levels in vivo is dependent on circadian regulation of eEF-2 activity. In contrast, rhythms in phosphorylated eEF-2 levels are not necessary for rhythms in accumulation of the clock protein FRQ, indicating that clock control of eEF-2 activity promotes rhythmic translation of specific mRNAs