Circadian clock control of tRNA synthetases in Neurospora crassa
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Background: In Neurospora crassa, the circadian clock controls rhythmic mRNA translation initiation through regulation of the eIF2 kinase CPC-3 (the homolog of yeast and mammalian GCN2). Active CPC-3 phosphorylates and inactivates eIF2, leading to higher phosphorylated eIF2 (P-eIF2) levels and reduced translation initiation during the subjective day. This daytime activation of CPC-3 is driven by its binding to uncharged tRNA, and uncharged tRNA levels peak during the day under control of the circadian clock. The daily rhythm in uncharged tRNA levels could arise from rhythmic amino acid levels or aminoacyl-tRNA synthetase (aaRSs) levels.Methods: To determine if and how the clock potentially controls rhythms in aspartyl-tRNA synthetase (AspRS) and glutaminyl-tRNA synthetase (GlnRS), both observed to be rhythmic in circadian genomic datasets, transcriptional and translational fusions to luciferase were generated. These luciferase reporter fusions were examined in wild type (WT), clock mutant frq, and clock-controlled transcription factor deletion strains.Results: Translational and transcriptional fusions of AspRS and GlnRS to luciferase confirmed that their protein and RNA levels are clock-controlled with peak levels at night. Clock-controlled transcription factor NCU00275was found to be necessary forrhythmic protein expression of AspRS and deletion of transcription factor ADV-1 decreased the amplitude of GlnRS rhythms.Conclusions: These data support a model whereby coordinate clock control of select aaRSs drives rhythms in uncharged tRNAs, leading to rhythmic CPC-3 activation, and rhythms in translation of specific mRNAs.
