The core body temperature of all mammals oscillates with the time of the day. However, direct molecular consequences of small, physiological changes in body temperature remain largely elusive. Here we show that body temperature cycles drive rhythmic SR protein phosphorylation to control an alternative splicing (AS) program. A temperature change of 1°C is sufficient to induce a concerted splicing switch in a large group of functionally related genes, rendering this splicing-based thermometer much more sensitive than previously described temperature-sensing mechanisms. AS of two exons in the 5' UTR of the TATA-box binding protein (Tbp) highlights the general impact of this mechanism, as it results in rhythmic TBP protein levels with implications for global gene expression in vivo. Together our data establish body temperature-driven AS as a core clock-independent oscillator in mammalian peripheral clocks.

Around the new or full moon, during a few specific hours surrounding low tide, millions of non-biting midges of the species C. marinus emerge from the sea to perform their nuptial dance. Adults live for only a few hours, during which they mate and oviposit. They must therefore emerge synchronously and-given that embryonic, larval and pupal development take place in the sea-at a time when the most extreme tides reliably expose the larval habitat. The lowest low tides occur predictably during specific days of the lunar month at a specific time of day. Consequently, adult emergence in C. marinus is under the control of circalunar and circadian clocks 1,2 . Notably, although the lowest low tides recur invariably at a given location, their timing differs between geographic locations 3 . Consequently, C. marinus strains from different locations (Extended Data Fig. 1a) show local adaptation in circadian and circalunar emergence times (Extended Data Fig. 1b, c). Crosses between the Jean and Por strains showed that the differences in circadian and circalunar timing are genetically determined 4,5 and largely explained by two circadian and two circalunar quantitative trait loci (QTLs) 6 . Studies on timing variation or chronotypes in animals and humans have often focused on candidate genes from the circadian transcriptiontranslational oscillator. In D. melanogaster, polymorphisms in the core circadian clock genes period, timeless and cryptochrome are associated with adaptive differences in temperature compensation 7 , photo-responsiveness of the circadian clock 8 and emergence rhythms 9 . While these studies offer insights into the evolution of known circadianclock molecules, genome-wide association studies 10,11 and other forward genetic approaches (reviewed in ref. 12) are essential to provide a comprehensive, unbiased assessment of natural timing variation, for instance underlying human sleep-phase disorders. While the adaptive nature of human chronotypes remains unclear, the chronotypes of C. marinus represent evolutionary adaptations to their habitat.Our study aimed to identify the genetic basis of C. marinus adaptation to its specific ecological 'timing niche' . In addition, the genetic dissection of adaptive natural variants of non-circadian rhythms 13 , as also present in C. marinus, may provide an entry point into their unknown molecular mechanisms.As a starting point for these analyses, we sequenced, assembled, mapped and annotated a C. marinus reference genome. The Clunio genome and QTLs for timingOur reference genome CLUMA_1.0 of the Jean laboratory strain contained 85.6 Mb of sequence (Table 1), close to the previous flowcytometry-based estimate of 95 Mb 6 , underlining that chironomids generally have small genomes [14][15][16] . The final assembly has a scaffold N50 of 1.9 Mb. Genome-wide genotyping of a mapping family with restriction-site associated DNA sequencing allowed 92% of the reference sequence to be consistently anchored along a genetic linkage map (Fig. 1a and Extended Data Fig. 2 Table 2). The C. ...

Highlights d CLK kinases are thermo-sensors reactive to subtle bodytemperature changes d Body temperature globally controls splicing and gene expression through CLKs d CLK homologs are evolutionarily adapted to diverse body/ growth temperatures d Wide functionality of CLKs: from TSD in reptiles to circadian biology in mammals

The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 disnRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation.[Keywords: pre-mRNA splicing; RNA helicase structure and function; remodeling of RNA-protein complexes; spliceosome catalytic activation; X-ray crystallography] Supplemental material is available for this article.Received September 14, 2015; revised version accepted November 13, 2015.Splicing entails the removal of noncoding sequences (introns) from primary transcripts and the concomitant ligation of neighboring coding regions (exons). It is mediated by a highly dynamic, multimegadalton RNA protein (RNP) molecular machine, the spliceosome, which consists of five small nuclear RNPs (snRNPs; U1, U2, U4, U5, and U6 in the case of the major spliceosome) and numerous non-snRNPs (Wahl et al. 2009). For each round of splicing, a spliceosome is assembled de novo on a substrate by the stepwise recruitment of snRNPs and nonsnRNPs. After assembly of a precatalytic complex, the spliceosome is catalytically activated and carries out the two consecutive steps of a splicing reaction before it is disassembled and its subunits are recycled. Each assembly, activation, catalysis, and disassembly step involves profound rearrangements of the spliceosomal RNP interaction networks, mediated predominantly by eight conserved superfamily 2 (SF2) NTPases/RNA helicases (Staley and Guthrie 1998). The most extensive rearrangements occur during spliceosome activation. In the precatalytic spliceosome, U4 and U6 snRNPs form a disnRNP by base-pairing of their snRNAs and are associated with U5 snRNP via protein-protein interactions. During spliceosome activation, the U5 snRNP-specific Brr2 helicase unwinds the U4/U6 ...

The circadian clock drives daily rhythms in gene expression to control metabolism, behavior, and physiology; while the underlying transcriptional feedback loops are well defined, the impact of alternative splicing on circadian biology remains poorly understood. Here we describe a robust circadian and light-inducible splicing switch that changes the reading frame of the mouse mRNA encoding U2-auxiliary-factor 26 (U2AF26). This results in translation far into the 3' UTR, generating a C terminus with homology to the Drosophila clock regulator TIMELESS. This new U2AF26 variant destabilizes PERIOD1 protein, and U2AF26-deficient mice show nearly arrhythmic PERIOD1 protein levels and broad defects in circadian mRNA expression in peripheral clocks. At the behavioral level, these mice display increased phase advance adaptation following experimental jet lag. These data suggest light-induced U2af26 alternative splicing to be a buffering mechanism that limits PERIOD1 induction, thus stabilizing the circadian clock against abnormal changes in light:dark conditions.