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Tissue Factor Produced by the Endocrine Cells of the Islets of Langerhans Is Associated With a Negative Outcome of Clinical Islet Transplantation Helena Johansson 1 , Agneta Lukinius 2 , Lisa Moberg 1 , Torbjörn Lundgren 3 , Christian Berne 4 , Aksel Foss 5 , Marie Felldin 6 , Ragnar Källen 7 , Kaija Salmela 8 , Annika Tibell 3 , Gunnar Tufveson 9 , Kristina Nilsson Ekdahl 1 10 , Graciela Elgue 1 , Olle Korsgren 1 and Bo Nilsson 1 1 Department of Radiology, Oncology and Clinical Immunology, Division of Clinical Immunology, The Rudbeck Laboratory, University Hospital, Uppsala, Sweden 2 Department of Genetics and Pathology, Division of Pathology, The Rudbeck Laboratory, University Hospital, Uppsala, Sweden 3 Department of Transplantation Surgery, Karolinska University Hospital, Stockholm, Sweden 4 Department of Medical Sciences, Division of Medicine, University Hospital, Uppsala, Sweden 5 Department of Transplantation Surgery, Rikshospitalet, Oslo, Norway 6 Department of Transplantation, University Hospital, Gothenburg, Sweden 7 Department of Nephrology and Transplantation, University Hospital, Malmö, Sweden 8 Division of Transplantation, Surgical Hospital, Helsinki University, Helsinki, Finland 9 Department of Surgical Sciences, Division of Transplantation Surgery, University Hospital, Uppsala, Sweden 10 Department of Chemistry and Biomedical Sciences, University of Kalmar, Kalmar, Sweden Address correspondencereprint requests to Helena Johansson, The Rudbeck Laboratory, University Hospital, 751 85 Uppsala, Sweden. E-mail: helena.johansson{at}klinimm.uu.se Abstract There are strong indications that only a small fraction of grafts successfully engraft in clinical islet transplantation. One explanation may be the instant blood-mediated inflammatory reaction (IBMIR) elicited by tissue factor, which is produced by the endocrine cells. In the present study, we show that islets intended for islet transplantation produce tissue factor in both the transmembrane and the alternatively spliced form and that the membrane-bound form is released as microparticles often associated with both insulin and glucagon granules. A low–molecular mass factor VIIa (FVIIa) inhibitor that indirectly blocks both forms of tissue factor was shown in vitro to be a promising drug to eliminate the IBMIR. Thrombin-antithrombin complex (TAT) and FVIIa-antithrombin complex (FVIIa-AT) were measured in nine patients who together received 20 infusions of isolated human islets. Both the TAT and FVIIa-AT complexes increased rapidly within 15–60 min after infusion. When the initial TAT and FVIIa-AT levels were plotted against the increase in C-peptide concentration after 7 days, patients with an initially strong IBMIR showed no significant increase in insulin synthesis after 7 days. In conclusion, tissue factor present in both the islets and the culture medium and elicits IBMIR, which affects the function of the transplanted islets. FVIIa, factor VIIa, FVIIa-AT, FVIIa-antithrombin complex IBMIR, instant blood-mediated inflammatory reaction mAbs, monoclonal antibodies TAT, thrombin-antithrombin complex Footnotes The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted February 22, 2005. Received May 4, 2004. DIABETES

Key Points Alternative splicing explains how a single gene can generate more than one mRNA transcript, thus expanding the complexity of the proteome. During normal development, a large number of alternative splicing changes occur, and it is now apparent that these transitions between alternatively spliced isoforms contribute to the acquisition of adult tissue functions and identity. Individual splicing changes are coordinated during development, establishing splicing networks. As a result of recent progress, we now better understand the mechanisms that coordinate alternative splicing networks and the roles of these networks in cell differentiation, organ development and tissue homeostasis. Alternative splicing expands the complexity of the proteome by generating multiple transcript isoforms from a single gene. Numerous alternative splicing events occur during cell differentiation and tissue maturation, suggesting that alternative splicing supports proper development. Recent studies shed light on how alternative splicing and its coordination contribute to organ development and tissue homeostasis. Alternative splicing of eukaryotic transcripts is a mechanism that enables cells to generate vast protein diversity from a limited number of genes. The mechanisms and outcomes of alternative splicing of individual transcripts are relatively well understood, and recent efforts have been directed towards studying splicing networks. It has become apparent that coordinated splicing networks regulate tissue and organ development, and that alternative splicing has important physiological functions in different developmental processes in humans.

Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 ( PRPF31 -mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31 +/− mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31 +/− mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical – basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies. Mutations in pre-mRNA processing factors cause autosomal dominant retinitis pigmentosa. Here the authors provide insights into the pathophysiological mechanisms underlying non-syndromic retinal disease caused by heterozygous mutations in genes encoding ubiquitously expressed splicing factors.

The insulin receptor (IR) gene undergoes differential splicing that generates two IR isoforms, IR-A and IR-B. The physiological roles of IR isoforms are incompletely understood and appear to be determined by their different binding affinities for insulin-like growth factors (IGFs), particularly for IGF-2. Predominant roles of IR-A in prenatal growth and development and of IR-B in metabolic regulation are well established. However, emerging evidence indicates that the differential expression of IR isoforms may also help explain the diversification of insulin and IGF signaling and actions in various organs and tissues by involving not only different ligand-binding affinities but also different membrane partitioning and trafficking and possibly different abilities to interact with a variety of molecular partners. Of note, dysregulation of the IR-A/IR-B ratio is associated with insulin resistance, aging, and increased proliferative activity of normal and neoplastic tissues and appears to sustain detrimental effects. This review discusses novel information that has generated remarkable progress in our understanding of the physiology of IR isoforms and their role in disease. We also focus on novel IR ligands and modulators that should now be considered as an important strategy for better and safer treatment of diabetes and cancer and possibly other IR-related diseases.We discuss recent work on the physiology of IR isoforms and their role in disease including new findings on IR ligands and modulators that are relevant to the treatment of diabetes and other disorders.

Key Points Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) of proteins that may also contain structured domains mediate crucial signalling processes in eukaryotic cells. Disorder is advantageous in cell signalling because disordered sequences have the potential to bind to multiple partners, often using different structures. Disordered regions are relatively accessible, often contain multiple binding motifs and are frequently the sites for post-translational modification, an important mediator of the control of signalling pathways. Disordered proteins have central roles in the formation of higher-order signalling assemblies and in the operation of circadian clocks. Intrinsically disordered proteins (IDPs) are key components of the cellular signalling machinery. Their flexible conformation enables them to interact with different partners and to participate in the assembly of signalling complexes and membrane-less organelles; this leads to different cellular outcomes. Post-translational modification of IDPs and alternative splicing add complexity to regulatory networks. Intrinsically disordered proteins (IDPs) are important components of the cellular signalling machinery, allowing the same polypeptide to undertake different interactions with different consequences. IDPs are subject to combinatorial post-translational modifications and alternative splicing, adding complexity to regulatory networks and providing a mechanism for tissue-specific signalling. These proteins participate in the assembly of signalling complexes and in the dynamic self-assembly of membrane-less nuclear and cytoplasmic organelles. Experimental, computational and bioinformatic analyses combine to identify and characterize disordered regions of proteins, leading to a greater appreciation of their widespread roles in biological processes.

Circular RNAs (circRNAs) have emerged as key regulators of human cancers, yet their modes of action in gastric cancer (GC) remain largely unknown. Here, we identified circURI1 back-spliced from exons 3 and 4 of unconventional prefoldin RPB5 interactor 1 (URI1) from circRNA profiling of five-paired human gastric and the corresponding nontumor adjacent specimens (paraGC). CircURI1 exhibits the significantly higher expression in GC compared with paraGC and inhibitory effects on cell migration and invasion in vitro and GC metastasis in vivo. Mechanistically, circURI1 directly interacts with heterogeneous nuclear ribonucleoprotein M (hnRNPM) to modulate alternative splicing of genes, involved in the process of cell migration, thus suppressing GC metastasis. Collectively, our study expands the current knowledge regarding the molecular mechanism of circRNA-mediated cancer metastasis via modulating alternative splicing.

Splicing varies across brain regions, but the single-cell resolution of regional variation is unclear. We present a single-cell investigation of differential isoform expression (DIE) between brain regions using single-cell long-read sequencing in mouse hippocampus and prefrontal cortex in 45 cell types at postnatal day 7 ( www.isoformAtlas.com ). Isoform tests for DIE show better performance than exon tests. We detect hundreds of DIE events traceable to cell types, often corresponding to functionally distinct protein isoforms. Mostly, one cell type is responsible for brain-region specific DIE. However, for fewer genes, multiple cell types influence DIE. Thus, regional identity can, although rarely, override cell-type specificity. Cell types indigenous to one anatomic structure display distinctive DIE, e.g. the choroid plexus epithelium manifests distinct transcription-start-site usage. Spatial transcriptomics and long-read sequencing yield a spatially resolved splicing map. Our methods quantify isoform expression with cell-type and spatial resolution and it contributes to further our understanding of how the brain integrates molecular and cellular complexity. Alternative RNA splicing varies across the brain. Its mapping at single cell resolution is unclear. Here, the authors provide a spatial and single-cell splicing atlas reporting brain region- and cell type-specific expression of different isoforms in the postnatal mouse brain.

Summary Plant can acquire tolerance to environmental stresses via transcriptome reprogramming at transcriptional and alternative splicing (AS) levels. However, how AS coordinates with transcriptional regulation to contribute to abiotic stresses responses is still ambiguous. In this study, we performed genome‐wide analyses of AS responses to drought stress (DS), heat stress (HS) and their combination (HD) in wheat seedlings, and further compared them with transcriptional responses. In total, we found 200, 3576 and 4056 genes exhibiting significant AS pattern changes in response to DS, HS and HD, respectively, and combined drought and heat stress can induce specific AS compared with individual one. In addition, wheat homeologous genes exhibited differential AS responses under stress conditions that more AS events occurred on B subgenome than on A and D genomes. Comparison of genes regulated at AS and transcriptional levels showed that only 12% of DS‐induced AS genes were subjected to transcriptional regulation, whereas the proportion increased to ~40% under HS and HD. Functional enrichment analysis revealed that abiotic stress‐responsive pathways tended to be highly overrepresented among these overlapped genes under HS and HD. Thus, we proposed that transcriptional regulation may play a major role in response to DS, which coordinates with AS regulation to contribute to HS and HD tolerance in wheat.

Alternative splicing is an essential post-transcriptional regulatory mechanism that can impact mRNA stability and protein diversity of eukaryotic genomes. Although numerous forms of stress-responsive alternative splicing have been identified in model plants, a large-scale study of alternative splicing dynamics under abiotic stress conditions in cassava has not been conducted. Here, we report the parallel employment of isoform-Seq, ssRNA-Seq, and Degradome- Seq to investigate the diversity, abundance, and fate of alternatively spliced isoforms in response to cold and drought stress. We identified 38 164 alternative splicing events, among which 3292 and 1025 events were significantly regulated by cold and drought stress, respectively. Intron retention was the most abundant subtype of alternative splicing. Global analysis of splicing regulators revealed that the number of their alternatively spliced isoforms and the corresponding abundance were specifically modulated by cold stress. We found that 58.5% of cold-regulated alternative splicing events introduced a premature termination codon into the transcripts, and 77.6% of differential alternative splicing events were detected by Degradome-Seq. Our data reveal that cold intensely affects both quantitative and qualitative aspects of gene expression via alternative splicing pathways, and advances our understanding of the high complexity and specificity of gene regulation in response to abiotic stresses.

Key Points Dysregulation of splicing, an emerging cause of many neurological disorders, affects all aspects of neurobiology from neurogenesis to synaptic function. The interplay between polypyrimidine tract binding protein 1 (PTBP1), serine/arginine repetitive matrix protein 4 (SRRM4), miR-124 and the REST (repressor element 1-silencing transcription factor) complex constitutes an important genetic programme underlying neuronal cell fate commitment. Neuronal migration during development requires cell surface receptor isoforms, the splicing of which is controlled by neuro-oncological ventral antigen 2 (NOVA2) and RNA-binding protein fox-1 homologue 2 (RBFOX2). The PTBP, SRRM4 and NOVA proteins orchestrate developmental synapse formation. Many splicing regulators, including KH domain-containing, RNA-binding, signal transduction-associated (KHDRBS) proteins, NOVA2 and muscleblind-like 2 (MBNL2), modulate synaptic transmission and plasticity. RBFOX1 and neuronal ELAV-like (nELAVL) regulate neuronal excitability. Understanding the biological roles of neuronal splicing regulators is challenged by extensive target sets, pleiotropic phenotypes and partial redundancy of paralogous regulators. Mutations in both protein regulators and core RNAs of the spliceosome can lead to neurodegenerative disorders. Alternative splicing is a key regulatory step in gene expression that affects all aspects of neuronal development and function. In this Review, Vuong and colleagues survey recent genetic studies of splicing regulators and the diverse parts they play in the mammalian nervous system. Alternative precursor-mRNA splicing is a key mechanism for regulating gene expression in mammals and is controlled by specialized RNA-binding proteins. The misregulation of splicing is implicated in multiple neurological disorders. We describe recent mouse genetic studies of alternative splicing that reveal its critical role in both neuronal development and the function of mature neurons. We discuss the challenges in understanding the extensive genetic programmes controlled by proteins that regulate splicing, both during development and in the adult brain.