period and timeless mRNA Splicing Profiles under Natural Conditions in Drosophila melanogaster

Previous analysis of Drosophila circadian behavior under natural conditions has revealed a number of novel and unexpected features. Here we focus on the oscillations of per and tim mRNAs and their posttranscriptional regulation and observe significant differences in molecular cycling under laboratory and natural conditions. In particular, robust per mRNA cycling from fly heads is limited to the summers, whereas tim RNA cycling is observed throughout the year. When both transcripts do cycle, their phases are similar, except for the very warmest summer months. We also study the natural splicing profiles of per and tim transcripts and observe a clear relationship between temperature and splicing. In natural conditions, we confirm the relationship between accumulation of the perspliced variant, low temperature, and the onset of the evening component of locomotor activity, first described in laboratory conditions. Intriguingly, in the case of tim splicing, we detect the opposite relationship, with timspliced expression increasing at higher temperatures. A first characterization of the 4 different TIM protein isoforms (resulting from the combination of the natural N-terminus length polymorphism and the C-terminus alternative splicing) using the 2-hybrid assay showed that the TIMunspliced isoforms have a stronger affinity for CRY, but not for PER, suggesting that the tim 3′ splicing could have physiological significance, possibly in temperature entrainment and/or adaptation to seasonal environments.