PRD-2 mediates clock-regulated perinuclear localization of clock gene RNAs within the circadian cycle of Neurospora

• Published in: Proceedings of the National Academy of Sciences - PNAS 2022-08, Vol.119 (31), p.e2203078119
• Main Authors: Bartholomai, Bradley M, Gladfelter, Amy S, Loros, Jennifer J, Dunlap, Jay C
• Format: Article
• Language: English
• Subjects: Biological clocks; Biological Sciences; Casein; Casein kinase I; Circadian Clocks - genetics; Circadian rhythm; Circadian Rhythm - genetics; Circadian rhythms; Clock gene; CLOCK Proteins - genetics; CLOCK Proteins - metabolism; Clustering; Feedback loops; Fluorescence in situ hybridization; Fungal Proteins - genetics; Fungal Proteins - metabolism; In Situ Hybridization, Fluorescence; Kinases; Localization; Negative feedback; Neurospora; Neurospora crassa - genetics; Neurospora crassa - metabolism; Nuclei; Oscillators; Period 1 protein; Period Circadian Proteins - genetics; Period Circadian Proteins - metabolism; Proteins; Ribonucleic acid; RNA; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA-binding protein; Time of use; Transcription, Genetic; Translation
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.2203078119

The transcription-translation negative feedback loops underlying animal and fungal circadian clocks are remarkably similar in their molecular regulatory architecture and, although much is understood about their central mechanism, little is known about the spatiotemporal dynamics of the gene products involved. A common feature of these circadian oscillators is a significant temporal delay between rhythmic accumulation of clock messenger RNAs (mRNAs) encoding negative arm proteins, for example, in and in mammals, and the appearance of the clock protein complexes assembled from the proteins they encode. Here, we report use of single-molecule RNA fluorescence in situ hybridization (smFISH) to show that the fraction of nuclei actively transcribing the clock gene changes in a circadian manner, and that these mRNAs cycle in abundance with fewer than five transcripts per nucleus at any time. Spatial point patterning statistics reveal that is spatially clustered near nuclei in a time of day-dependent manner and that clustering requires an RNA-binding protein, PRD-2 (PERIOD-2), recently shown also to bind to mRNA encoding another core clock component, casein kinase 1. An intrinsically disordered protein, PRD-2 displays behavior in vivo and in vitro consistent with participation in biomolecular condensates. These data are consistent with a role for phase-separating RNA-binding proteins in spatiotemporally organizing clock mRNAs to facilitate local translation and assembly of clock protein complexes.