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.

Regulation of 3' splice site selection after step 1 of splicing by spliceosomal C* proteins.

Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3' splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA-mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3'ss. Cryo-electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3' region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3'ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3'ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3'ss choices.

ASO targeting RBM3 temperature-controlled poison exon splicing prevents neurodegeneration in vivo.

Neurodegenerative diseases are increasingly prevalent in the aging population, yet no disease-modifying treatments are currently available. Increasing the expression of the cold-shock protein RBM3 through therapeutic hypothermia is remarkably neuroprotective. However, systemic cooling poses a health risk, strongly limiting its clinical application. Selective upregulation of RBM3 at normothermia thus holds immense therapeutic potential. Here we identify a poison exon within the RBM3 gene that is solely responsible for its cold-induced expression. Genetic removal or antisense oligonucleotide (ASO)-mediated manipulation of this exon yields high RBM3 levels independent of cooling. Notably, a single administration of ASO to exclude the poison exon, using FDA-approved chemistry, results in long-lasting increased RBM3 expression in mouse brains. In prion-diseased mice, this treatment leads to remarkable neuroprotection, with prevention of neuronal loss and spongiosis despite high levels of disease-associated prion protein. Our promising results in mice support the possibility that RBM3-inducing ASOs might also deliver neuroprotection in humans in conditions ranging from acute brain injury to Alzheimer's disease.

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/).

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.

Structural and functional investigation of the human snRNP assembly factor AAR2 in complex with the RNase H-like domain of PRPF8.

Small nuclear ribonucleoprotein complexes (snRNPs) represent the main subunits of the spliceosome. While the assembly of the snRNP core particles has been well characterized, comparably little is known of the incorporation of snRNP-specific proteins and the mechanisms of snRNP recycling. U5 snRNP assembly in yeast requires binding of the the Aar2 protein to Prp8p as a placeholder to preclude premature assembly of the SNRNP200 helicase, but the role of the human AAR2 homolog has not yet been investigated in detail. Here, a crystal structure of human AAR2 in complex with the RNase H-like domain of the U5-specific PRPF8 (PRP8F RH) is reported, revealing a significantly different interaction between the two proteins compared with that in yeast. Based on the structure of the AAR2-PRPF8 RH complex, the importance of the interacting regions and residues was probed and AAR2 variants were designed that failed to stably bind PRPF8 in vitro. Protein-interaction studies of AAR2 with U5 proteins using size-exclusion chromatography reveal similarities and marked differences in the interaction patterns compared with yeast Aar2p and imply phosphorylation-dependent regulation of AAR2 reminiscent of that in yeast. It is found that in vitro AAR2 seems to lock PRPF8 RH in a conformation that is only compatible with the first transesterification step of the splicing reaction and blocks a conformational switch to the step 2-like, Mg2+-coordinated conformation that is likely during U5 snRNP biogenesis. These findings extend the picture of AAR2 PRP8 interaction from yeast to humans and indicate a function for AAR2 in the spliceosomal assembly process beyond its role as an SNRNP200 placeholder in yeast.

Recruitment of a splicing factor to the nuclear lamina for its inactivation.

Precursor messenger RNA splicing is a highly regulated process, mediated by a complex RNA-protein machinery, the spliceosome, that encompasses several hundred proteins and five small nuclear RNAs in humans. Emerging evidence suggests that the spatial organization of splicing factors and their spatio-temporal dynamics participate in the regulation of splicing. So far, methods to manipulate the spatial distribution of splicing factors in a temporally defined manner in living cells are missing. Here, we describe such an approach that takes advantage of a reversible chemical dimerizer, and outline the requirements for efficient, reversible re-localization of splicing factors to selected sub-nuclear compartments. In a proof-of-principle study, the partial re-localization of the PRPF38A protein to the nuclear lamina in HEK293T cells induced a moderate increase in intron retention. Our approach allows fast and reversible re-localization of splicing factors, has few side effects and can be applied to many splicing factors by fusion of a protein tag through genome engineering. Apart from the systematic analysis of the spatio-temporal aspects of splicing regulation, the approach has a large potential for the fast induction and reversal of splicing switches and can reveal mechanisms of splicing regulation in native nuclear environments.

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.

A multi-factor trafficking site on the spliceosome remodeling enzyme BRR2 recruits C9ORF78 to regulate alternative splicing.

The intrinsically unstructured C9ORF78 protein was detected in spliceosomes but its role in splicing is presently unclear. We find that C9ORF78 tightly interacts with the spliceosome remodeling factor, BRR2, in vitro. Affinity purification/mass spectrometry and RNA UV-crosslinking analyses identify additional C9ORF78 interactors in spliceosomes. Cryogenic electron microscopy structures reveal how C9ORF78 and the spliceosomal B complex protein, FBP21, wrap around the C-terminal helicase cassette of BRR2 in a mutually exclusive manner. Knock-down of C9ORF78 leads to alternative NAGNAG 3'-splice site usage and exon skipping, the latter dependent on BRR2. Inspection of spliceosome structures shows that C9ORF78 could contact several detected spliceosome interactors when bound to BRR2, including the suggested 3'-splice site regulating helicase, PRPF22. Together, our data establish C9ORF78 as a late-stage splicing regulatory protein that takes advantage of a multi-factor trafficking site on BRR2, providing one explanation for suggested roles of BRR2 during splicing catalysis and alternative splicing.

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.

Splicing-accessible coding 3'UTRs control protein stability and interaction networks.

BACKGROUND: 3'-Untranslated regions (3'UTRs) play crucial roles in mRNA metabolism, such as by controlling mRNA stability, translation efficiency, and localization. Intriguingly, in some genes the 3'UTR is longer than their coding regions, pointing to additional, unknown functions. Here, we describe a protein-coding function of 3'UTRs upon frameshift-inducing alternative splicing in more than 10% of human and mouse protein-coding genes. RESULTS: 3'UTR-encoded amino acid sequences show an enrichment of PxxP motifs and lead to interactome rewiring. Furthermore, an elevated proline content increases protein disorder and reduces protein stability, thus allowing splicing-controlled regulation of protein half-life. This could also act as a surveillance mechanism for erroneous skipping of penultimate exons resulting in transcripts that escape nonsense mediated decay. The impact of frameshift-inducing alternative splicing on disease development is emphasized by a retinitis pigmentosa-causing mutation leading to translation of a 3'UTR-encoded, proline-rich, destabilized frameshift-protein with altered protein-protein interactions. CONCLUSIONS: We describe a widespread, evolutionarily conserved mechanism that enriches the mammalian proteome, controls protein expression and protein-protein interactions, and has important implications for the discovery of novel, potentially disease-relevant protein variants.

Srsf10 and the minor spliceosome control tissue-specific and dynamic SR protein expression.

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.

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.

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.

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.

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.

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.

Activation-Dependent TRAF3 Exon 8 Alternative Splicing Is Controlled by CELF2 and hnRNP C Binding to an Upstream Intronic Element.

Cell-type-specific and inducible alternative splicing has a fundamental impact on regulating gene expression and cellular function in a variety of settings, including activation and differentiation. We have recently shown that activation-induced skipping of TRAF3 exon 8 activates noncanonical NF-κB signaling upon T cell stimulation, but the regulatory basis for this splicing event remains unknown. Here we identify cis- and trans-regulatory elements rendering this splicing switch activation dependent and cell type specific. The cis-acting element is located 340 to 440 nucleotides upstream of the regulated exon and acts in a distance-dependent manner, since altering the location reduces its activity. A small interfering RNA screen, followed by cross-link immunoprecipitation and mutational analyses, identified CELF2 and hnRNP C as trans-acting factors that directly bind the regulatory sequence and together mediate increased exon skipping in activated T cells. CELF2 expression levels correlate with TRAF3 exon skipping in several model systems, suggesting that CELF2 is the decisive factor, with hnRNP C being necessary but not sufficient. These data suggest an interplay between CELF2 and hnRNP C as the mechanistic basis for activation-dependent alternative splicing of TRAF3 exon 8 and additional exons and uncover an intronic splicing silencer whose full activity depends on the precise location more than 300 nucleotides upstream of the regulated exon.

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.