Tumour necrosis factor-a (TNF-a), a proinflammatory cytokine, is a potent negative regulator of adipocyte differentiation. However, the mechanism of TNF-a-mediated antiadipogenesis remains incompletely understood. In this study, we first confirm that TNF-a inhibits adipogenesis of 3T3-L1 preadipocytes by preventing the early induction of the adipogenic transcription factors peroxisome proliferator-activated receptor-c (PPARc) and CCAAT/enhancer binding protein-a (C/EBPa). This suppression coincides with enhanced expression of several reported mediators of antiadipogenesis that are also targets of the Wnt/b-catenin/T-cell factor 4 (TCF4) pathway. Indeed, we found that TNF-a enhanced TCF4-dependent transcriptional activity during early antiadipogenesis, and promoted the stabilisation of b-catenin throughout antiadipogenesis. We analysed the effect of TNF-a on adipogenesis in 3T3-L1 cells in which b-catenin/TCF signalling was impaired, either via stable knockdown of b-catenin, or by overexpression of dominant-negative TCF4 (dnTCF4). The knockdown of b-catenin enhanced the adipogenic potential of 3T3-L1 preadipocytes and attenuated TNF-a-induced antiadipogenesis. However, b-catenin knockdown also promoted TNF-a-induced apoptosis in these cells. In contrast, overexpression of dnTCF4 prevented TNF-a-induced antiadipogenesis but showed no apparent effect on cell survival. Finally, we show that TNF-a-induced antiadipogenesis and stabilisation of b-catenin requires a functional death domain of TNF-a receptor 1 (TNFR1). Taken together these data suggest that TNFR1-mediated death domain signals can inhibit adipogenesis via a b-catenin/TCF4-dependent pathway.

No abstract

Signal-induced alternative splicing of the CD45 gene in human T cells is essential for proper immune function. Skipping of the CD45 variable exons is controlled, in large part, by the recruitment of PSF to the pre-mRNA substrate upon T cell activation; however, the signaling cascade leading to exon exclusion has remained elusive. Here we demonstrate that in resting T cells PSF is directly phosphorylated by GSK3 thus promoting interaction of PSF with TRAP150 which prevents PSF from binding CD45 pre-mRNA. Upon T cell activation, reduced GSK3 activity leads to reduced PSF phosphorylation, releasing PSF from TRAP150 and allowing it to bind CD45 splicing regulatory elements and repress exon inclusion. Our data place two new players, GSK3 and TRAP150, in the complex network that regulates CD45 alternative splicing and demonstrate a new paradigm for signal transduction from the cell surface to the RNA processing machinery through the multi-functional protein PSF.

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.

MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4+ T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation.

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. ...

Splicing regulatory proteins often have distinct activities when bound to exons versus introns. However, less clear is whether variables besides location can influence activity. HnRNP L binds to a motif present in both CD45 variable exons 4 and 5 to affect their coordinate repression. Here we show that, in contrast to its direct repression of exon 4, hnRNP L represses exon 5 by countering the activity of a neighboring splicing enhancer. In the absence of the enhancer hnRNP L unexpectedly activates exon inclusion. As the splice sites flanking exon 4 and 5 are distinct, we directly examined the effect of varying splice site strength on the mechanism of hnRNP L function. Remarkably, binding of hnRNP L to an exon represses strong splice sites but enhances weak splice sites. A model in which hnRNP L stabilizes snRNP binding can explain both effects in a manner determined by the inherent snRNP-substrate affinity.

The formation and execution of a productive immune response requires the maturation of competent T cells and a robust change in cellular activity upon antigen challenge. Such changes in cellular function depend on regulated alterations to protein expression. Previous research has focused on defining transcriptional changes that regulate protein expression during T-cell maturation and antigen stimulation. Here, we globally analyze another critical process in gene regulation during T-cell stimulation, alternative splicing. Specifically, we use RNA-seq profiling to identify 178 exons in 168 genes that exhibit robust changes in inclusion in response to stimulation of a human T-cell line. Supporting an important role for the global coordination of alternative splicing following T-cell stimulation, these signal-responsive exons are significantly enriched in genes with functional annotations specifically related to immune response. The vast majority of these genes also exhibit differential alternative splicing between naive and activated primary T cells. Comparison of the responsiveness of splicing to various stimuli in the cultured and primary T cells further reveals at least three distinct networks of signal-induced alternative splicing events. Importantly, we find that each regulatory network is specifically associated with distinct sequence features, suggesting that they are controlled by independent regulatory mechanisms. These results thus provide a basis for elucidating mechanisms of signal pathway-specific regulation of alternative splicing during T-cell stimulation.