A single intron (Intron P) in the Drosophila timeless gene functions as an RNA-based molecular timer that regulates circadian period length through inefficient post-transcriptional splicing, which retains ~50% of tim mRNAs in the nucleus, thereby controlling the timing and levels of TIM protein accumulation; CRISPR-mediated deletion of Intron P abolishes nuclear retention and shortens the circadian period to ~22 hours, while insertion of Intron P into heterologous reporters is sufficient to promote nuclear retention in both Drosophila and human cells, establishing splicing kinetics as a conserved regulatory mechanism for temporal gene expression.

: Circadian clocks regulate daily rhythms in all eukaryotes through ∼24-hour transcriptional-translational feedback loops driven by clock proteins. However, the molecular mechanisms that set the 24-hour period remain poorly defined. Here, using single-molecule imaging and nascent RNA sequencing, we uncover an unexpected RNA-based molecular timer: a single intron in the Drosophila timeless (tim) gene that regulates circadian period length by controlling mRNA localization. Strikingly, we find that ∼50% of tim mRNAs are localized to the nucleus due to inefficient post-transcriptional splicing of a single intron (which we named intron P), in contrast to other core clock transcripts that localize predominantly to the cytoplasm. CRISPR-mediated removal of intron P abolishes nuclear retention of tim transcripts, leading to accelerated TIM protein accumulation and a shortened ∼22-hour period with reduced rhythmic robustness. Remarkably, insertion of intron P alone into heterologous reporters is sufficient to promote nuclear retention in both Drosophila and human cells, acting as a conserved checkpoint that withholds transcripts in the nucleus until splicing is complete. Finally, we identify three RNA-binding proteins, two repressors (Hrb27C and Squid) and one activator (Qkr58E-2, a Sam68 homolog), that modulate intron P splicing in a rheostat-like manner. Together, these findings establish tim intron P as the first intron-based molecular timer in circadian clocks and reveal splicing kinetics as a critical regulatory layer in temporal gene expression programs, with broad implications for other processes such as development and immunity.