Mechanisms of Circadian Clock Control of Rhythmic Translation in Neurospora crassa

The circadian clock in Neurospora crassa regulates daily rhythms in the phosphorylation and daytime inactivation of the conserved translation initiation factor eIF2α. Clock control of eIF2α activity is responsible for the rhythmic translation of ~15% of mRNAs. Cycling phosphorylated eIF2α levels require rhythmic activation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2). However, how the clock controls the activity of CPC-3 is not known, and this information is critical to determine the mechanisms underlying rhythmic protein synthesis. To be activated, CPC-3 forms a complex with GCN1, which helps to bring uncharged tRNAs to the tRNA binding domain on CPC-3. In Saccharomyces cerevisiae, activation of GCN2 under stress conditions requires direct interaction of GCN1 and GCN2 with ribosomes. Furthermore, CPC-3 and GCN1 levels are clock-controlled in N. crassa. Based on these data, I hypothesized that N. crassa CPC-3 and GCN1 rhythmically interact with the ribosome, and that this interaction is necessary for rhythmic CPC-3 activity and eIF2α-controlled translation initiation. To test this hypothesis, the interaction of CPC-3 and GCN1 with ribosomes was examined in WT and the clock mutant Δfrq. Ribosomes were pelleted from cultures grown in constant dark (DD) and harvested every 4 hours in a circadian time course. I found that CPC-3 and GCN1 interact with monosomes and polysomes, and that the interaction is clock-regulated with peak levels during the subjective day. We showed previously that rhythms in uncharged tRNA levels, and rhythms in CPC-3 activity are abolished in a valyl tRNA synthetase temperature sensitive mutant (un-3ts). The rhythmic interaction of CPC-3 and GCN1 with ribosomes was abolished in the un-3ts mutant, suggesting that rhythmic levels of uncharged tRNA drives the rhythmic interaction of CPC-3 and GCN1 with ribosomes. I found that disrupting the interaction between GCN1 and uncharged tRNA in the absence of GCN20, affects rhythmic CPC-3 activity. Taken together, these data support that clock regulation of rhythms in uncharged tRNA levels and rhythms in the interaction between CPC-3 and GCN1 with ribosomes are necessary for rhythmic CPC-3 activity that leads to rhythms in the translation of target mRNAs.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Microbiology

Grantor

Texas A&M University

Year dc:date.issued

2024

Author dc:creator

• Preh, Ebimobowei Olugbenga

Advisor dc:contributor.advisor

• Bell-Pedersen, Deborah

Committee members dc:contributor.committeemember

• Ebbole, Daniel
• Sachs, Matthew
• Merlin, Christine

• Biology, General
• Biology, Molecular
• Biology, Genetics

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/1969.1/1588902

OAI record oai:identifier

https://oaktrust.library.tamu.edu/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Aoaktrust.library.tamu.edu%3A1969.1%2F1588902