Post-transcriptional splicing of timeless drives ~24-hour circadian rhythms in Drosophila

Circadian clocks drive ~24-hour rhythms in physiology and behavior through precisely timed gene expression cycles1-4. Here, we reveal an unexpected role for intron splicing in tuning the ~24-hour circadian period in Drosophila. Using RNA Fluorescent In Situ Hybridization and RNA-sequencing, we demonstrate that ~50% of timeless (tim) mRNAs—a core clock transcript—remain nuclear throughout the circadian cycle due to retention of a specific intron, termed intron P. Unlike other tim introns, spliced during transcription, intron P—lacking a canonical branch point—is spliced inefficiently post-transcriptionally near nuclear speckles, thereby limiting tim mRNA export and TIM protein synthesis. CRISPR deletion of intron P results in all tim mRNAs being fully spliced and exported, accelerating TIM production and shortening the circadian period to ~22.5 hours. Inserting intron P into reporter genes promotes their nuclear retention in both Drosophila neurons and human U2OS cells, which is reversed by adding a canonical branch point sequence. A genetic screen identified the KH-domain RNA-binding protein Qkr58E-2 (a Sam68 homolog5) as an intron P splicing activator, and HnRNP proteins Hrb27c and Squid6 as repressors. Together, these findings uncover a novel regulatory role for intron splicing dynamics in circadian timing and further suggest that introns have the potential to act as critical regulatory elements in other temporally regulated biological processes, including developmental transitions and stress responses. Overall design: polyA RNA sequencing of Drosophila melanogaster clock neurons in control and Prp3-RNAi knockdown conditions. Cells were isolated by fluorescence-activated cell sorting (FACS) using Clk-GAL4>UAS-GFP-NLS line developed in the lab. GFP-positive and DAPI-negative cells were sorted to be clock neurons.