Unveiling the Impact of the Negative Arm of the Circadian Clock on Output in Neurospora crassa

Abstract

Circadian rhythms are cellular oscillations that tune biology to Earth's 24-hour light/dark cycle, aiding in the anticipation of environmental changes. These rhythms are maintained by an evolutionarily conserved transcriptional/translational negative feedback loop, referred to as the core circadian clock. In this core clock, positive arm proteins act to transcriptionally activate negative arm proteins that act back on the positive arm to halt the negative arm's activation before degradation and re-initiation, thus setting the period of the clock. Most research into circadian regulation on output has focused on transcriptional activation by the positive arm proteins. However, recently published work has demonstrated that there are many oscillating proteins for which the corresponding mRNA is not rhythmic. This has led to the theory that the post-translational regulation of circadian output exists, which we hypothesize is imparted by the negative arm proteins. In the model organism Neurospora crassa, the negative arm includes FREQUENCY (FRQ). FRQ has been computationally and experimentally proven to be an intrinsically disordered protein (IDP). Aside from lacking a predetermined three-dimensional structure, IDPs play key roles in cellular signaling and regulation due, in part, to the transient but highly specific and varied interacting partners they have. These attributes make FRQ a prime candidate to impart circadian regulation post-translationally. The aim of this study is to characterize protein complexes, centered around FRQ, that change in a time specific manor to determine the impact these interactions could impart on circadian regulatory pathways. To this end, Neurospora samples where crosslinked and purified through co-immunoprecipitation before protein identification through liquid chromatography mass spectrometry. Initial results suggest that FRQ plays a role in regulation on metabolism, the cell cycle, and a variety of other functions through novel protein interactions.