The unfolded protein response regulates ER exit sites via SNRPB-dependent RNA splicing and contributes to bone development.

Splicing and endoplasmic reticulum (ER)-proteostasis are two key processes that ultimately regulate the functional proteins that are produced by a cell. However, the extent to which these processes interact remains poorly understood. Here, we identify SNRPB and other components of the Sm-ring, as targets of the unfolded protein response and novel regulators of export from the ER. Mechanistically, The Sm-ring regulates the splicing of components of the ER export machinery, including Sec16A, a component of ER exit sites. Loss of function of SNRPB is causally linked to cerebro-costo-mandibular syndrome (CCMS), a genetic disease characterized by bone defects. We show that heterozygous deletion of SNRPB in mice resulted in bone defects reminiscent of CCMS and that knockdown of SNRPB delays the trafficking of type-I collagen. Silencing SNRPB inhibited osteogenesis in vitro, which could be rescued by overexpression of Sec16A. This rescue indicates that the role of SNRPB in osteogenesis is linked to its effects on ER-export. Finally, we show that SNRPB is a target for the unfolded protein response, which supports a mechanistic link between the spliceosome and ER-proteostasis. Our work highlights components of the Sm-ring as a novel node in the proteostasis network, shedding light on CCMS pathophysiology.

A Conserved Kinase-Based Body-Temperature Sensor Globally Controls Alternative Splicing and Gene Expression.

Homeothermic organisms maintain their core body temperature in a narrow, tightly controlled range. Whether and how subtle circadian oscillations or disease-associated changes in core body temperature are sensed and integrated in gene expression programs remain elusive. Furthermore, a thermo-sensor capable of sensing the small temperature differentials leading to temperature-dependent sex determination (TSD) in poikilothermic reptiles has not been identified. Here, we show that the activity of CDC-like kinases (CLKs) is highly responsive to physiological temperature changes, which is conferred by structural rearrangements within the kinase activation segment. Lower body temperature activates CLKs resulting in strongly increased phosphorylation of SR proteins in vitro and in vivo. This globally controls temperature-dependent alternative splicing and gene expression, with wide implications in circadian, tissue-specific, and disease-associated settings. This temperature sensor is conserved across evolution and adapted to growth temperatures of diverse poikilotherms. The dynamic temperature range of reptilian CLK homologs suggests a role in TSD.

Body Temperature Cycles Control Rhythmic Alternative Splicing in Mammals.

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.

Thermoregulated transcriptomics: the molecular basis and biological significance of temperature-dependent alternative splicing.

Temperature-dependent alternative splicing (AS) is a crucial mechanism for organisms to adapt to varying environmental temperatures. In mammals, even slight fluctuations in body temperature are sufficient to drive significant AS changes in a concerted manner. This dynamic regulation allows organisms to finely tune gene expression and protein isoform diversity in response to temperature cues, ensuring proper cellular function and physiological adaptation. Understanding the molecular mechanisms underlying temperature-dependent AS thus provides valuable insights into the intricate interplay between environmental stimuli and gene expression regulation. In this review, we provide an overview of recent advances in understanding temperature-regulated AS across various biological processes and systems. We will discuss the machinery sensing and translating temperature cues into changed AS patterns, the adaptation of the splicing regulatory machinery to extreme temperatures, the role of temperature-dependent AS in shaping the transcriptome, functional implications and the development of potential therapeutics targeting temperature-sensitive AS pathways.

Srsf1 and Elavl1 act antagonistically on neuronal fate choice in the developing neocortex by controlling TrkC receptor isoform expression.

The seat of higher-order cognitive abilities in mammals, the neocortex, is a complex structure, organized in several layers. The different subtypes of principal neurons are distributed in precise ratios and at specific positions in these layers and are generated by the same neural progenitor cells (NPCs), steered by a spatially and temporally specified combination of molecular cues that are incompletely understood. Recently, we discovered that an alternatively spliced isoform of the TrkC receptor lacking the kinase domain, TrkC-T1, is a determinant of the corticofugal projection neuron (CFuPN) fate. Here, we show that the finely tuned balance between TrkC-T1 and the better known, kinase domain-containing isoform, TrkC-TK+, is cell type-specific in the developing cortex and established through the antagonistic actions of two RNA-binding proteins, Srsf1 and Elavl1. Moreover, our data show that Srsf1 promotes the CFuPN fate and Elavl1 promotes the callosal projection neuron (CPN) fate in vivo via regulating the distinct ratios of TrkC-T1 to TrkC-TK+. Taken together, we connect spatio-temporal expression of Srsf1 and Elavl1 in the developing neocortex with the regulation of TrkC alternative splicing and transcript stability and neuronal fate choice, thus adding to the mechanistic and functional understanding of alternative splicing in vivo.

Body temperature variation controls pre-mRNA processing and transcription of antiviral genes and SARS-CoV-2 replication.

Antiviral innate immunity represents the first defense against invading viruses and is key to control viral infections, including SARS-CoV-2. Body temperature is an omnipresent variable but was neglected when addressing host defense mechanisms and susceptibility to SARS-CoV-2 infection. Here, we show that increasing temperature in a 1.5°C window, between 36.5 and 38°C, strongly increases the expression of genes in two branches of antiviral immunity, nitric oxide production and type I interferon response. We show that alternative splicing coupled to nonsense-mediated decay decreases STAT2 expression in colder conditions and suggest that increased STAT2 expression at elevated temperature induces the expression of diverse antiviral genes and SARS-CoV-2 restriction factors. This cascade is activated in a remarkably narrow temperature range below febrile temperature, which reflects individual, circadian and age-dependent variation. We suggest that decreased body temperature with aging contributes to reduced expression of antiviral genes in older individuals. Using cell culture and in vivo models, we show that higher body temperature correlates with reduced SARS-CoV-2 replication, which may affect the different vulnerability of children versus seniors toward severe SARS-CoV-2 infection. Altogether, our data connect body temperature and pre-mRNA processing to provide new mechanistic insight into the regulation of antiviral innate immunity.

One pipeline to predict them all? On the prediction of alternative splicing from RNA-Seq data.

RNA-Seq has become the standard approach to quantify and compare gene expression and alternative splicing in different conditions. In many cases the limiting factor is not the sequencing itself but the bioinformatic analysis. A variety of software tools exist that predict alternative splicing patterns from RNA-Seq data, but surprisingly, a systematic comparison of the predictions obtained from different pipelines has not been performed. Here we compare results from frequently used bioinformatic tools using a high-quality RNA-Seq dataset. We show that there is little overlap in the splicing changes predicted by different tools and that GO-term analysis of the splicing changes predicted by the individual targets yields very different results. Validation of bioinformatic predictions by RT-PCR suggest a high number of false positives in the splicing changes predicated by each pipeline, which probably dominates GO-term analysis. The validation rate is strongly increased for targets predicted by several tools, offering a strategy to reduce false positives. Based on these results we offer some guidelines that may contribute to make alternative splicing predictions more reliable and may thus increase the impact of conclusions drawn from RNA-Seq studies. Furthermore, we created rmappet, a nextflow pipeline that performs alternative splicing analysis using rMATS and Whippet with subsequent overlapping of the results, enabling robust splicing analysis with only one command (https://github.com/didrikolofsson/rmappet/).

The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation.

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 di-snRNP 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.

Alternative splicing coupled mRNA decay shapes the temperature-dependent transcriptome.

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.

The genomic basis of circadian and circalunar timing adaptations in a midge.

Organisms use endogenous clocks to anticipate regular environmental cycles, such as days and tides. Natural variants resulting in differently timed behaviour or physiology, known as chronotypes in humans, have not been well characterized at the molecular level. We sequenced the genome of Clunio marinus, a marine midge whose reproduction is timed by circadian and circalunar clocks. Midges from different locations show strain-specific genetic timing adaptations. We examined genetic variation in five C. marinus strains from different locations and mapped quantitative trait loci for circalunar and circadian chronotypes. The region most strongly associated with circadian chronotypes generates strain-specific differences in the abundance of calcium/calmodulin-dependent kinase II.1 (CaMKII.1) splice variants. As equivalent variants were shown to alter CaMKII activity in Drosophila melanogaster, and C. marinus (Cma)-CaMKII.1 increases the transcriptional activity of the dimer of the circadian proteins Cma-CLOCK and Cma-CYCLE, we suggest that modulation of alternative splicing is a mechanism for natural adaptation in circadian timing.

Rhythmic U2af26 alternative splicing controls PERIOD1 stability and the circadian clock in mice.

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.

A Snu114-GTP-Prp8 module forms a relay station for efficient splicing in yeast.

The single G protein of the spliceosome, Snu114, has been proposed to facilitate splicing as a molecular motor or as a regulatory G protein. However, available structures of spliceosomal complexes show Snu114 in the same GTP-bound state, and presently no Snu114 GTPase-regulatory protein is known. We determined a crystal structure of Snu114 with a Snu114-binding region of the Prp8 protein, in which Snu114 again adopts the same GTP-bound conformation seen in spliceosomes. Snu114 and the Snu114-Prp8 complex co-purified with endogenous GTP. Snu114 exhibited weak, intrinsic GTPase activity that was abolished by the Prp8 Snu114-binding region. Exchange of GTP-contacting residues in Snu114, or of Prp8 residues lining the Snu114 GTP-binding pocket, led to temperature-sensitive yeast growth and affected the same set of splicing events in vivo. Consistent with dynamic Snu114-mediated protein interactions during splicing, our results suggest that the Snu114-GTP-Prp8 module serves as a relay station during spliceosome activation and disassembly, but that GTPase activity may be dispensable for splicing.

The zinc finger domains in U2AF26 and U2AF35 have diverse functionalities including a role in controlling translation.

Recent work has associated point mutations in both zinc fingers (ZnF) of the spliceosome component U2AF35 with malignant transformation. However, surprisingly little is known about the functionality of the U2AF35 ZnF domains in general. Here we have analysed key functionalities of the ZnF domains of mammalian U2AF35 and its paralog U2AF26. Both ZnFs are required for splicing regulation, whereas only ZnF2 controls protein stability and contributes to the interaction with U2AF65. These features are confirmed in a naturally occurring splice variant of U2AF26 lacking ZnF2, that is strongly induced upon activation of primary mouse T cells and localized in the cytoplasm. Using Ribo-Seq in a model T cell line we provide evidence for a role of U2AF26 in activating cytoplasmic steps in gene expression, notably translation. Consistently, an MS2 tethering assay shows that cytoplasmic U2AF26/35 increase translation when localized to the 5'UTR of a model mRNA. This regulation is partially dependent on ZnF1 thus providing a connection between a core splicing factor, the ZnF domains and the regulation of translation. Altogether, our work reveals unexpected functions of U2AF26/35 and their ZnF domains, thereby contributing to a better understanding of their role and regulation in mammalian cells.

Temperature-controlled Rhythmic Gene Expression in Endothermic Mammals: All Diurnal Rhythms are Equal, but Some are Circadian.

The circadian clock is a cell autonomous oscillator that controls many aspects of physiology through generating rhythmic gene expression in a time of day dependent manner. In addition, in endothermic mammals body temperature cycles contribute to rhythmic gene expression. These body temperature-controlled rhythms are hard to distinguish from classic circadian rhythms if analyzed in vivo in endothermic organisms. However, they do not fulfill all criteria of being circadian if analyzed in cell culture or in conditions where body temperature of an endothermic organism can be manipulated. Here we review and compare these characteristics, discuss the core clock independent mechanism of temperature-controlled alternative splicing and highlight the requirement of double-checking rhythms that appear circadian within an endothermic organism in a system that allows temperature manipulation.

Characterization of cis-acting elements that control oscillating alternative splicing.

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.

Post-transcriptional control of the mammalian circadian clock: implications for health and disease.

Many aspects of human physiology and behavior display rhythmicity with a period of approximately 24 h. Rhythmic changes are controlled by an endogenous time keeper, the circadian clock, and include sleep-wake cycles, physical and mental performance capability, blood pressure, and body temperature. Consequently, many diseases, such as metabolic, sleep, autoimmune and mental disorders and cancer, are connected to the circadian rhythm. The development of therapies that take circadian biology into account is thus a promising strategy to improve treatments of diverse disorders, ranging from allergic syndromes to cancer. Circadian alteration of body functions and behavior are, at the molecular level, controlled and mediated by widespread changes in gene expression that happen in anticipation of predictably changing requirements during the day. At the core of the molecular clockwork is a well-studied transcription-translation negative feedback loop. However, evidence is emerging that additional post-transcriptional, RNA-based mechanisms are required to maintain proper clock function. Here, we will discuss recent work implicating regulated mRNA stability, translation and alternative splicing in the control of the mammalian circadian clock, and its role in health and disease.

Activation-induced tumor necrosis factor receptor-associated factor 3 (Traf3) alternative splicing controls the noncanonical nuclear factor κB pathway and chemokine expression in human T cells.

The noncanonical nuclear factor κB (ncNFκB) pathway regulates the expression of chemokines required for secondary lymphoid organ formation and thus plays a pivotal role in adaptive immunity. Whereas ncNFκB signaling has been well described in stromal cells and B cells, its role and regulation in T cells remain largely unexplored. ncNFκB activity critically depends on the upstream NFκB-inducing kinase (NIK). NIK expression is negatively regulated by the full-length isoform of TNF receptor-associated factor 3 (Traf3) as formation of a NIK-Traf3-Traf2 complex targets NIK for degradation. Here we show that T cell-specific and activation-dependent alternative splicing generates a Traf3 isoform lacking exon 8 (Traf3DE8) that, in contrast to the full-length protein, activates ncNFκB signaling. Traf3DE8 disrupts the NIK-Traf3-Traf2 complex and allows accumulation of NIK to initiate ncNFκB signaling in activated T cells. ncNFκB activity results in expression of several chemokines, among them B cell chemoattractant (CxCL13), both in a model T cell line and in primary human CD4(+) T cells. Because CxCL13 plays an important role in B cell migration and activation, our data suggest an involvement and provide a mechanistic basis for Traf3 alternative splicing and ncNFκB activation in contributing to T cell-dependent adaptive immunity.

HnRNP L and L-like cooperate in multiple-exon regulation of CD45 alternative splicing.

CD45 encodes a trans-membrane protein-tyrosine phosphatase expressed in diverse cells of the immune system. By combinatorial use of three variable exons 4-6, isoforms are generated that differ in their extracellular domain, thereby modulating phosphatase activity and immune response. Alternative splicing of these CD45 exons involves two heterogeneous ribonucleoproteins, hnRNP L and its cell-type specific paralog hnRNP L-like (LL). To address the complex combinatorial splicing of exons 4-6, we investigated hnRNP L/LL protein expression in human B-cells in relation to CD45 splicing patterns, applying RNA-Seq. In addition, mutational and RNA-binding analyses were carried out in HeLa cells. We conclude that hnRNP LL functions as the major CD45 splicing repressor, with two CA elements in exon 6 as its primary target. In exon 4, one element is targeted by both hnRNP L and LL. In contrast, exon 5 was never repressed on its own and only co-regulated with exons 4 and 6. Stable L/LL interaction requires CD45 RNA, specifically exons 4 and 6. We propose a novel model of combinatorial alternative splicing: HnRNP L and LL cooperate on the CD45 pre-mRNA, bridging exons 4 and 6 and looping out exon 5, thereby achieving full repression of the three variable exons.

The nuclear GYF protein CD2BP2/U5-52K is required for T cell homeostasis.

The question whether interference with the ubiquitous splicing machinery can lead to cell-type specific perturbation of cellular function is addressed here by T cell specific ablation of the general U5 snRNP assembly factor CD2BP2/U5-52K. This protein defines the family of nuclear GYF domain containing proteins that are ubiquitously expressed in eukaryotes with essential functions ascribed to early embryogenesis and organ function. Abrogating CD2BP2/U5-52K in T cells, allows us to delineate the consequences of splicing machinery interferences for T cell development and function. Increased T cell lymphopenia and T cell death are observed upon depletion of CD2BP2/U5-52K. A substantial increase in exon skipping coincides with the observed defect in the proliferation/differentiation balance in the absence of CD2BP2/U5-52K. Prominently, skipping of exon 7 in Mdm4 is observed, coinciding with upregulation of pro-apoptotic gene expression profiles upon CD2BP2/U5-52K depletion. Furthermore, we observe enhanced sensitivity of naïve T cells compared to memory T cells to changes in CD2BP2/U5-52K levels, indicating that depletion of this general splicing factor leads to modulation of T cell homeostasis. Given the recent structural characterization of the U5 snRNP and the crosslinking mass spectrometry data given here, design of inhibitors of the U5 snRNP conceivably offers new ways to manipulate T cell function in settings of disease.

Branch point strength controls species-specific CAMK2B alternative splicing and regulates LTP.

Regulation and functionality of species-specific alternative splicing has remained enigmatic to the present date. Calcium/calmodulin-dependent protein kinase IIß (CaMKIIß) is expressed in several splice variants and plays a key role in learning and memory. Here, we identify and characterize several primate-specific CAMK2B splice isoforms, which show altered kinetic properties and changes in substrate specificity. Furthermore, we demonstrate that primate-specific CAMK2B alternative splicing is achieved through branch point weakening during evolution. We show that reducing branch point and splice site strengths during evolution globally renders constitutive exons alternative, thus providing novel mechanistic insight into cis-directed species-specific alternative splicing regulation. Using CRISPR/Cas9, we introduce a weaker, human branch point sequence into the mouse genome, resulting in strongly altered Camk2b splicing in the brains of mutant mice. We observe a strong impairment of long-term potentiation in CA3-CA1 synapses of mutant mice, thus connecting branch point-controlled CAMK2B alternative splicing with a fundamental function in learning and memory.