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.

Minor and major spliceosomes control splicing of distinct intron types and are thought to act largely independent of one another. SR proteins are essential splicing regulators mostly connected to the major spliceosome. Here, we show that Srsf10 expression is controlled through an autoregulated minor intron, tightly correlating Srsf10 with minor spliceosome abundance across different tissues and differentiation stages in mammals. Surprisingly, all other SR proteins also correlate with the minor spliceosome and Srsf10, and abolishing Srsf10 autoregulation by Crispr/Cas9-mediated deletion of the autoregulatory exon induces expression of all SR proteins in a human cell line. Our data thus reveal extensive crosstalk and a global impact of the minor spliceosome on major intron splicing.

Mammalian body temperature oscillates with the time of the day and is altered in diverse pathological conditions. We recently identified a body temperature‐sensitive thermometer‐like kinase, which alters SR protein phosphorylation and thereby globally controls alternative splicing (AS). AS can generate unproductive variants which are recognized and degraded by diverse mRNA decay pathways—including nonsense‐mediated decay (NMD). Here we show extensive coupling of body temperature‐controlled AS to mRNA decay, leading to global control of temperature‐dependent gene expression (GE). Temperature‐controlled, decay‐inducing splicing events are evolutionarily conserved and pervasively found within RNA‐binding proteins, including most SR proteins. AS‐coupled poison exon inclusion is essential for rhythmic GE of SR proteins and has a global role in establishing temperature‐dependent rhythmic GE profiles, both in mammals under circadian body temperature cycles and in plants in response to ambient temperature changes. Together, these data identify body temperature‐driven AS‐coupled mRNA decay as an evolutionary ancient, core clock‐independent mechanism to generate rhythmic GE.

Alternative splicing (AS) in response to changing external conditions often requires alterations in the ability of sequence-specific RNA-binding proteins to bind to cis-acting sequences in their target pre-mRNA. While daily oscillations in AS events have been described in several organisms, cis-acting sequences that control time of the day-dependent AS remain largely elusive. Here we define cis-regulatory RNA elements that control body-temperature driven rhythmic AS using the mouse U2af26 gene as a model system. We identify a complex network of cis-regulatory sequences that regulate AS of U2af26, and show that the activity of two enhancer elements is necessary for oscillating AS. A minigene comprising these U2af26 regions recapitulates rhythmic splicing of the endogenous gene, which is controlled through temperature-regulated SR protein phosphorylation. Mutagenesis of the minigene delineates the cis-acting enhancer element for SRSF2 within exon 6 to single nucleotide resolution and reveals that the combined activity of SRSF2 and SRSF7 is required for oscillating U2af26 AS. By combining RNA-Seq with an siRNA screen and individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP), we identify a complex network of SR proteins that globally controls temperature-dependent rhythmic AS, with the direction of splicing depending on the position of the cis-acting elements. Together, we provide detailed insights into the sequence requirements that allow trans-acting factors to generate daily rhythms in AS.

2Mammalian body temperature oscillates with the time of the day and is altered in diverse 3 pathological conditions. We recently identified a body temperature-sensitive thermometer-like 4 kinase, which alters SR protein phosphorylation and thereby globally controls alternative splicing 5 (AS). AS can generate mRNA variants containing premature termination codons, which are 6 degraded by nonsense-mediated decay (NMD). Here we show extensive coupling of body 7 temperature-controlled AS to NMD, leading to global control of temperature-dependent gene 8 expression (GE). Temperature-controlled NMD-inducing splicing events are evolutionarily 9 conserved and pervasively found within RNA-binding proteins, including most SR proteins. NMD-10 inducing exons are essential for rhythmic GE of SR proteins and have a global role in establishing 11 temperature-dependent rhythmic GE profiles, both, in mammals under circadian body temperature 12 cycles and in plants in response to ambient temperature changes. Together, these data identify 13 body temperature-driven AS-NMD as an evolutionary ancient, core clock-independent mechanism 14 to generate rhythmic GE. 15 16 Keywords 17Alternative splicing / circadian clock / NMD / SR proteins / temperature 18