Deciphering the Role of mRNA Nuclear Localization in Circadian Rhythms

University Of Michigan At Ann Arbor, Ann Arbor MI

Investigators

Abstract

Abstract Circadian clocks align our physiological functions with Earth's day-night cycles through rhythmic gene expression. While the genetic framework of circadian clocks has been well characterized, the mechanisms responsible for the crucial long-time delays within circadian feedback loops, necessary for establishing a 24-hour periodicity, remain poorly understood. While the importance of clock protein degradation in generating temporal delays was recognized over two decades ago, the roles of subcellular localization and RNA metabolism regulation in contributing to these delays have yet to be explored. To address this critical gap, our lab has recently adapted single-molecule RNA-FISH and a machine-learning-based analysis to visualize and quantify individual clock mRNAs in Drosophila clock neurons. Using this technique, we made an unexpected discovery that the mRNAs of timeless (tim), a core clock gene, are localized to not only the cytoplasm but also to the nucleus, specifically near the nuclear speckles. These nuclear speckles are RNP (ribonucleoprotein) condensates enriched for splicing factors, essential for mRNA processing. To identify the sequences responsible for the nuclear localization of tim mRNAs, we examined the splicing of tim introns in the clock neurons of Drosophila. Our studies have revealed an intriguing pattern in the splicing of tim introns: intron 5 is consistently retained in almost all nuclear tim mRNAs, unlike other tim introns, which are spliced at the gene locus concurrently with transcription. This distinctive retention of intron 5 suggests it is spliced post-transcriptionally, which could lead to a delay in the export of tim mRNA from the nucleus to the cytoplasm, resulting in a delayed accumulation of TIM protein. Further, a novel transgenic fly line with tim intron 5 deletion resulted in shortened ~22-h circadian rhythms, suggesting that regulated post-transcriptional splicing of this intron is critical for introducing delays in the circadian feedback loops, which is essential for ~24-h circadian rhythms. Next, we found that clock neuron specific knockdown of Precursor RNA Processing 3 (Prp3) protein, a U4/U6.U5 tri-snRNP splicing factor, caused a majority of tim mRNAs to be localized to the nucleus due to the failure to splice out tim intron 5 specifically, leading to very low levels of TIM protein and completely arrhythmic behavior. This study provides the first evidence, within any model system, that the post-transcriptional splicing of a specific intron within a core clock gene plays a critical role in generating essential temporal delays in ~24-h circadian clocks. In this grant, we aim to: i) decipher the sequences that are required and sufficient for the nuclear localization of tim mRNAs, and ii) determine the molecular mechanism by which Prp proteins regulate the post-transcriptional splicing of tim intron 5 and affect circadian rhythms. Our experiments will reveal novel mechanisms governing post-transcriptional splicing and mRNA nuclear localization, elucidating their precise role in regulating circadian rhythms and providing new insights into gene regulation broadly in development and disease.