Clock Control of Ribosome Composition and Activity: the Potential Role of the Ribosome-Associated Protein, Zuotin, in Neurospora crassa

Abstract

Daily circadian rhythms are under the control of an endogenous timekeeper, the circadian clock, which confers an adaptive advantage to organisms by providing the ability to prepare for daily environmental changes and to temporally organize cellular processes. The impact of the circadian clock on rhythmic gene expression is widespread; the levels of at least half of eukaryotic transcripts are controlled by the clock. Recent work in the Bell-Pedersen lab demonstrated that the clock also plays a role in regulating rhythmic mRNA translation; however, how the clock controls rhythmic translation is only just beginning to be understood. Using mass spectrometry of intact ribosomes over a circadian time course, our lab found that several Neurospora crassa ribosomal proteins and one ribosome interacting protein, Zuotin, exhibit rhythmic ribosome occupancy. Once thought to be homogenous cellular structures, we now know that these cellular machines can vary in their composition and function. To investigate if the clock regulates ribosome heterogeneity and if ribosome heterogeneity impacts mRNA translation, I tested the hypothesis that the circadian clock regulates ribosome composition and activity through rhythmic association of Zuotin with ribosomes in N. crassa. Zuotin deletion mutants were generated to explore Zuotin’s role in growth and the clock in N. crassa. Race tube assays of these mutants revealed a potential role for Zuotin in maintaining cell growth. A V5-tagged ZUO strain was generated to monitor rhythms in its interaction with ribosomes; however, ZUO::V5 protein levels were not rhythmic over a circadian time course. Additionally, pelleted ribosomes of ZUO::V5 in WT and ∆el31 strains showed significant difference in ZUO’s interaction with the ribosome, suggesting that while a ZUO-eL31 is not required, eL31 may potentially stabilize ZUO in N. crassa.