In the unfolded protein response (UPR), stress in the endoplasmic reticulum (ER) activates a large transcriptional program to increase ER folding capacity. During the budding yeast UPR, Ire1 excises an intron from the HAC1 mRNA and the exon products of cleavage are ligated, and the translated protein induces hundreds of stress-response genes. Using cells with mutations in RNA repair and decay enzymes, we show that phosphorylation of two different HAC1 splicing intermediates is required for their degradation by the 5'→3' exonuclease Xrn1 to enact opposing effects on the UPR. We also found that ligated but 2'-phosphorylated HAC1 mRNA is cleaved, yielding a decay intermediate with both 5'- and 2'-phosphates at its 5'-end that inhibit 5'→3' decay and suggesting that Ire1 degrades incompletely processed HAC1. These decay events expand the scope of RNA-based regulation in the budding yeast UPR and have implications for the control of the metazoan UPR.

Circular RNAs (circRNAs) represent an abundant and conserved entity of non-coding RNAs; however, the principles of biogenesis are currently not fully understood. Here, we identify two factors, splicing factor proline/glutamine rich (SFPQ) and non-POU domain-containing octamer-binding protein (NONO), to be enriched around circRNA loci. We observe a subclass of circRNAs, coined DALI circRNAs, with distal inverted Alu elements and long flanking introns to be highly deregulated upon SFPQ knockdown. Moreover, SFPQ depletion leads to increased intron retention with concomitant induction of cryptic splicing, premature transcription termination, and polyadenylation, particularly prevalent for long introns. Aberrant splicing in the upstream and downstream regions of circRNA producing exons are critical for shaping the circRNAome, and specifically, we identify missplicing in the immediate upstream region to be a conserved driver of circRNA biogenesis. Collectively, our data show that SFPQ plays an important role in maintaining intron integrity by ensuring accurate splicing of long introns, and disclose novel features governing Alu-independent circRNA production.

Prp3 is an essential U4/U6 di-snRNP-associated protein whose functions and molecular mechanisms in pre-mRNA splicing are presently poorly understood. We show by structural and biochemical analyses that Prp3 contains a bipartite U4/U6 di-snRNA-binding region comprising an expanded ferredoxin-like fold, which recognizes a 3'-overhang of U6 snRNA, and a preceding peptide, which binds U4/U6 stem II. Phylogenetic analyses revealed that the single-stranded RNA-binding domain is exclusively found in Prp3 orthologs, thus qualifying as a spliceosome-specific RNA interaction module. The composite double-stranded/single-stranded RNA-binding region assembles cooperatively with Snu13 and Prp31 on U4/U6 di-snRNAs and inhibits Brr2-mediated U4/U6 di-snRNA unwinding in vitro. RNP-disrupting mutations in Prp3 lead to U4/U6•U5 tri-snRNP assembly and splicing defects in vivo. Our results reveal how Prp3 acts as an important bridge between U4/U6 and U5 in the tri-snRNP and comparison with a Prp24-U6 snRNA recycling complex suggests how Prp3 may be involved in U4/U6 reassembly after splicing.

We show that the splicing regulator PTBP2 controls a genetic program essential for neuronal maturation. Depletion of PTBP2 in developing mouse cortex leads to degeneration of these tissues over the first three postnatal weeks, a time when the normal cortex expands and develops mature circuits. Cultured Ptbp2(-/-) neurons exhibit the same initial viability as wild type, with proper neurite outgrowth and marker expression. However, these mutant cells subsequently fail to mature and die after a week in culture. Transcriptome-wide analyses identify many exons that share a pattern of mis-regulation in the mutant brains, where isoforms normally found in adults are precociously expressed in the developing embryo. These transcripts encode proteins affecting neurite growth, pre- and post-synaptic assembly, and synaptic transmission. Our results define a new genetic regulatory program, where PTBP2 acts to temporarily repress expression of adult protein isoforms until the final maturation of the neuron. DOI: http://dx.doi.org/10.7554/eLife.01201.001.

Tissue- and cell-type-specific regulators of alternative splicing (AS) are essential components of posttranscriptional gene regulation, necessary for normal cellular function, patterning, and development. Mice with ablation of Epithelial splicing regulatory protein (Esrp1) develop cleft lip and palate. Loss of both Esrp1 and its paralog Esrp2 results in widespread developmental defects with broad implications to human disease. Deletion of the Esrps in the epidermis revealed their requirement for establishing a proper skin barrier, a primary function of epithelial cells comprising the epidermis. We profiled the global Esrp-mediated splicing regulatory program in epidermis, which revealed large-scale programs of epithelial cell-type-specific splicing required for epithelial cell functions. These mice represent a valuable model for evaluating the essential role for AS in development and function of epithelial cells, which play essential roles in tissue homeostasis in numerous organs, and provide a genetic tool to evaluate important functional properties of epithelial-specific splice variants in vivo.

The Drosophila polyadenosine RNA binding protein Nab2, which is orthologous to a human protein lost in a form of inherited intellectual disability, controls adult locomotion, axon projection, dendritic arborization, and memory through a largely undefined set of target RNAs. Here, we show a specific role for Nab2 in regulating splicing of ~150 exons/introns in the head transcriptome and focus on retention of a male-specific exon in the sex determination factor Sex-lethal (Sxl) that is enriched in female neurons. Previous studies have revealed that this splicing event is regulated in females by N6-methyladenosine (m6A) modification by the Mettl3 complex. At a molecular level, Nab2 associates with Sxl pre-mRNA in neurons and limits Sxl m6A methylation at specific sites. In parallel, reducing expression of the Mettl3, Mettl3 complex components, or the m6A reader Ythdc1 rescues mutant phenotypes in Nab2 flies. Overall, these data identify Nab2 as an inhibitor of m6A methylation and imply significant overlap between Nab2 and Mettl3 regulated RNAs in neuronal tissue.

Dysregulated pre-mRNA splicing and metabolism are two hallmarks of MYC-driven cancers. Pharmacological inhibition of both processes has been extensively investigated as potential therapeutic avenues in preclinical and clinical studies. However, how pre-mRNA splicing and metabolism are orchestrated in response to oncogenic stress and therapies is poorly understood. Here, we demonstrate that jumonji domain containing 6, arginine demethylase, and lysine hydroxylase, JMJD6, acts as a hub connecting splicing and metabolism in MYC-driven human neuroblastoma. JMJD6 cooperates with MYC in cellular transformation of murine neural crest cells by physically interacting with RNA binding proteins involved in pre-mRNA splicing and protein homeostasis. Notably, JMJD6 controls the alternative splicing of two isoforms of glutaminase (GLS), namely kidney-type glutaminase (KGA) and glutaminase C (GAC), which are rate-limiting enzymes of glutaminolysis in the central carbon metabolism in neuroblastoma. Further, we show that JMJD6 is correlated with the anti-cancer activity of indisulam, a 'molecular glue' that degrades splicing factor RBM39, which complexes with JMJD6. The indisulam-mediated cancer cell killing is at least partly dependent on the glutamine-related metabolic pathway mediated by JMJD6. Our findings reveal a cancer-promoting metabolic program is associated with alternative pre-mRNA splicing through JMJD6, providing a rationale to target JMJD6 as a therapeutic avenue for treating MYC-driven cancers.

When activated by the accumulation of unfolded proteins in the endoplasmic reticulum, metazoan IRE1, the most evolutionarily conserved unfolded protein response (UPR) transducer, initiates unconventional splicing of XBP1 mRNA. Unspliced and spliced mRNA are translated to produce pXBP1(U) and pXBP1(S), respectively. pXBP1(S) functions as a potent transcription factor, whereas pXBP1(U) targets pXBP1(S) to degradation. In addition, activated IRE1 transmits two signaling outputs independent of XBP1, namely activation of the JNK pathway, which is initiated by binding of the adaptor TRAF2 to phosphorylated IRE1, and regulated IRE1-dependent decay (RIDD) of various mRNAs in a relatively nonspecific manner. Here, we conducted comprehensive and systematic genetic analyses of the IRE1-XBP1 branch of the UPR using medaka fish and found that the defects observed in XBP1-knockout or IRE1-knockout medaka were fully rescued by constitutive expression of pXBP1(S). Thus, the JNK and RIDD pathways are not required for the normal growth and development of medaka. The unfolded protein response sensor/transducer IRE1-mediated splicing of XBP1 mRNA encoding its active downstream transcription factor to maintain the homeostasis of the endoplasmic reticulum is sufficient for growth and development of medaka fish.

The endoplasmic reticulum (ER) protein folding capacity is balanced with the protein folding burden to prevent accumulation of un- or misfolded proteins. The ER membrane-resident kinase/RNase Ire1 maintains ER protein homeostasis through two fundamentally distinct processes. First, Ire1 can initiate a transcriptional response through a non-conventional mRNA splicing reaction to increase the ER folding capacity. Second, Ire1 can decrease the ER folding burden through selective mRNA decay. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, the two Ire1 functions have been evolutionarily separated. Here, we show that the respective Ire1 orthologs have become specialized for their functional outputs by divergence of their RNase specificities. In addition, RNA structural features separate the splicing substrates from the decay substrates. Using these insights, we engineered an S. pombe Ire1 cleavage substrate into a splicing substrate, which confers S. pombe with both Ire1 functional outputs.

Alternative pre-mRNA splicing expands the complexity of the transcriptome and controls isoform-specific gene expression. Whether alternative splicing contributes to metabolic regulation is largely unknown. Here we investigated the contribution of alternative splicing to the development of diet-induced obesity. We found that obesity-induced changes in adipocyte gene expression include alternative pre-mRNA splicing. Bioinformatics analysis associated part of this alternative splicing program with sequence specific NOVA splicing factors. This conclusion was confirmed by studies of mice with NOVA deficiency in adipocytes. Phenotypic analysis of the NOVA-deficient mice demonstrated increased adipose tissue thermogenesis and improved glycemia. We show that NOVA proteins mediate a splicing program that suppresses adipose tissue thermogenesis. Together, these data provide quantitative analysis of gene expression at exon-level resolution in obesity and identify a novel mechanism that contributes to the regulation of adipose tissue function and the maintenance of normal glycemia.

Alternative splicing occurs in the vast majority of human genes, giving rise to distinct mRNA and protein isoforms. We, and others, have previously identified hundreds of genes that change their isoform expression upon T cell activation via alternative splicing; however, how these changes link activation input with functional output remains largely unknown. Here, we investigate how costimulation of T cells through the CD28 receptor impacts alternative splicing in T cells activated through the T cell receptor (TCR, CD3) and find that while CD28 signaling alone has minimal impact on splicing, it enhances the extent of change for up to 20% of TCR-induced alternative splicing events. Interestingly, a set of CD28-enhanced splicing events occur within genes encoding key components of the apoptotic signaling pathway; namely caspase-9, Bax, and Bim. Using both CRISPR-edited cells and antisense oligos to force expression of specific isoforms, we show for all three of these genes that the isoform induced by CD3/CD28 costimulation promotes resistance to apoptosis, and that changes in all three genes together function combinatorially to further promote cell viability. Finally, we show that the JNK signaling pathway, induced downstream of CD3/CD28 costimulation, is required for each of these splicing events, further highlighting their co-regulation. Together, these findings demonstrate that alternative splicing is a key mechanism by which costimulation of CD28 promotes viability of activated T cells.

Viral infection induces the expression of numerous host genes that impact the outcome of infection. Here, we show that infection of human lung epithelial cells with influenza A virus (IAV) also induces a broad program of alternative splicing of host genes. Although these splicing-regulated genes are not enriched for canonical regulators of viral infection, we find that many of these genes do impact replication of IAV. Moreover, in several cases, specific inhibition of the IAV-induced splicing pattern also attenuates viral infection. We further show that approximately a quarter of the IAV-induced splicing events are regulated by hnRNP K, a host protein required for efficient splicing of the IAV M transcript in nuclear speckles. Finally, we find an increase in hnRNP K in nuclear speckles upon IAV infection, which may alter accessibility of hnRNP K for host transcripts thereby leading to a program of host splicing changes that promote IAV replication.

S-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events. Cells regulate intracellular SAM levels through intron detention of MAT2A, the only SAM synthetase expressed in most cells. The N6-adenosine methyltransferase METTL16 promotes splicing of the MAT2A detained intron by an unknown mechanism. Using an unbiased CRISPR knock-out screen, we identified CFIm25 (NUDT21) as a regulator of MAT2A intron detention and intracellular SAM levels. CFIm25 is a component of the cleavage factor Im (CFIm) complex that regulates poly(A) site selection, but we show it promotes MAT2A splicing independent of poly(A) site selection. CFIm25-mediated MAT2A splicing induction requires the RS domains of its binding partners, CFIm68 and CFIm59 as well as binding sites in the detained intron and 3´ UTR. These studies uncover mechanisms that regulate MAT2A intron detention and reveal a previously undescribed role for CFIm in splicing and SAM metabolism.

Circadian rhythms are generated by the cyclic transcription, translation, and degradation of clock gene products, including timeless (tim), but how the circadian clock senses and adapts to temperature changes is not completely understood. Here, we show that temperature dramatically changes the splicing pattern of tim in Drosophila. We found that at 18°C, TIM levels are low because of the induction of two cold-specific isoforms: tim-cold and tim-short and cold. At 29°C, another isoform, tim-medium, is upregulated. Isoform switching regulates the levels and activity of TIM as each isoform has a specific function. We found that tim-short and cold encodes a protein that rescues the behavioral defects of tim01 mutants, and that flies in which tim-short and cold is abrogated have abnormal locomotor activity. In addition, miRNA-mediated control limits the expression of some of these isoforms. Finally, data that we obtained using minigenes suggest that tim alternative splicing might act as a thermometer for the circadian clock.

Circadian rhythms are maintained by a cell-autonomous, transcriptional-translational feedback loop known as the molecular clock. While previous research suggests a role of the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomere filaments. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMSBmal1-/-) mice, we showed that knocking out skeletal muscle clock function alters titin isoform expression using RNAseq, liquid chromatography-mass spectrometry, and sodium dodecyl sulfate-vertical agarose gel electrophoresis. This alteration in titin's spring length resulted in sarcomere length heterogeneity. We demonstrate the direct link between altered titin splicing and sarcomere length in vitro using U7 snRNPs that truncate the region of titin altered in iMSBmal1-/- muscle. We identified a mechanism whereby the skeletal muscle clock regulates titin isoform expression through transcriptional regulation of Rbm20, a potent splicing regulator of titin. Lastly, we used an environmental model of circadian rhythm disruption and identified significant downregulation of Rbm20 expression. Our findings demonstrate the importance of the skeletal muscle circadian clock in maintaining titin isoform through regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many chronic diseases, our results highlight a novel pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies.

Insects are the only known animals in which sexual differentiation is controlled by sex-specific splicing. The doublesex transcription factor produces distinct male and female isoforms, which are both essential for sex-specific development. dsx splicing depends on transformer, which is also alternatively spliced such that functional Tra is only present in females. This pathway has evolved from an ancestral mechanism where dsx was independent of tra and expressed and required only in males. To reconstruct this transition, we examined three basal, hemimetabolous insect orders: Hemiptera, Phthiraptera, and Blattodea. We show that tra and dsx have distinct functions in these insects, reflecting different stages in the changeover from a transcription-based to a splicing-based mode of sexual differentiation. We propose that the canonical insect tra-dsx pathway evolved via merger between expanding dsx function (from males to both sexes) and narrowing tra function (from a general splicing factor to dedicated regulator of dsx).

The nonsense-mediated mRNA decay (NMD) pathway detects aberrant transcripts containing premature termination codons (PTCs) and regulates expression of 5-10% of non-aberrant human mRNAs. To date, most proteins involved in NMD have been identified by genetic screens in model organisms; however, the increased complexity of gene expression regulation in human cells suggests that additional proteins may participate in the human NMD pathway. To identify proteins required for NMD, we performed a genome-wide RNAi screen against >21,000 genes. Canonical members of the NMD pathway were highly enriched as top hits in the siRNA screen, along with numerous candidate NMD factors, including the conserved ICE1/KIAA0947 protein. RNAseq studies reveal that depletion of ICE1 globally enhances accumulation and stability of NMD-target mRNAs. Further, our data suggest that ICE1 uses a putative MIF4G domain to interact with exon junction complex (EJC) proteins and promotes the association of the NMD protein UPF3B with the EJC.

Self-splicing introns are mobile elements that have invaded a number of highly conserved genes in prokaryotic and organellar genomes. Here, we show that deletion of these selfish elements from the Saccharomyces cerevisiae mitochondrial genome is stressful to the host. A strain without mitochondrial introns displays hallmarks of the retrograde response, with altered mitochondrial morphology, gene expression and metabolism impacting growth and lifespan. Deletion of the complete suite of mitochondrial introns is phenocopied by overexpression of the splicing factor Mss116. We show that, in both cases, abnormally efficient transcript maturation results in excess levels of mature cob and cox1 host mRNA. Thus, inefficient splicing has become an integral part of normal mitochondrial gene expression. We propose that the persistence of S. cerevisiae self-splicing introns has been facilitated by an evolutionary lock-in event, where the host genome adapted to primordial invasion in a way that incidentally rendered subsequent intron loss deleterious.

To mount a protective response to infection while preventing hyperinflammation, gene expression in innate immune cells must be tightly regulated. Despite the importance of pre-mRNA splicing in shaping the proteome, its role in balancing immune outcomes remains understudied. Transcriptomic analysis of murine macrophage cell lines identified Serine/Arginine Rich Splicing factor 6 (SRSF6) as a gatekeeper of mitochondrial homeostasis. SRSF6-dependent orchestration of mitochondrial health is directed in large part by alternative splicing of the pro-apoptosis pore-forming protein BAX. Loss of SRSF6 promotes accumulation of BAX-κ, a variant that sensitizes macrophages to undergo cell death and triggers upregulation of interferon stimulated genes through cGAS sensing of cytosolic mitochondrial DNA. Upon pathogen sensing, macrophages regulate SRSF6 expression to control the liberation of immunogenic mtDNA and adjust the threshold for entry into programmed cell death. This work defines BAX alternative splicing by SRSF6 as a critical node not only in mitochondrial homeostasis but also in the macrophage's response to pathogens.

Drosophila syncytial nuclear divisions limit transcription unit size of early zygotic genes. As mitosis inhibits not only transcription, but also pre-mRNA splicing, we reasoned that constraints on splicing were likely to exist in the early embryo, being splicing avoidance a possible explanation why most early zygotic genes are intronless. We isolated two mutant alleles for a subunit of the NTC/Prp19 complexes, which specifically impaired pre-mRNA splicing of early zygotic but not maternally encoded transcripts. We hypothesized that the requirements for pre-mRNA splicing efficiency were likely to vary during development. Ectopic maternal expression of an early zygotic pre-mRNA was sufficient to suppress its splicing defects in the mutant background. Furthermore, a small early zygotic transcript with multiple introns was poorly spliced in wild-type embryos. Our findings demonstrate for the first time the existence of a developmental pre-requisite for highly efficient splicing during Drosophila early embryonic development and suggest in highly proliferative tissues a need for coordination between cell cycle and gene architecture to ensure correct gene expression and avoid abnormally processed transcripts. DOI: http://dx.doi.org/10.7554/eLife.02181.001.