Fibronectin extra domain a limits liver dysfunction and protects mice during acute inflammation.

Background and aim: The primary transcript of fibronectin (FN) undergoes alternative splicing to generate different isoforms, including FN containing the Extra Domain A (FN_EDA+), whose expression is regulated spatially and temporarily during developmental and disease conditions including acute inflammation. The role of FN_EDA+ during sepsis, however, remains elusive. Methods: Mice constitutively express the EDA domain of fibronectin (EDA+/+); lacking the FN EDA domain (EDA-/-) or with a conditional ablation of EDA + inclusion only in liver produced FN (alb-CRE+EDA floxed mice) thus expressing normal plasma FN were used. Systemic inflammation and sepsis were induced by either LPS injection (70 mg/kg) or by cecal ligation and puncture (CLP) Neutrophils isolated from septic patients were tested for neutrophil binding ability. Results: We observed that EDA+/+ were protected toward sepsis as compared to EDA-/- mice. Also alb-CRE+EDA floxed mice presented reduced survival, thus indicating a key role for EDA in protecting toward sepsis. This phenotype was associated with improved liver and spleen inflammatory profile. Ex vivo experiments showed that neutrophils bind to a larger extent to an FN_EDA + coated surface as compared to FN, thus potentially limiting their over-reactivity. Conclusions: Our study demonstrates that the inclusion of the EDA domain in fibronectin dampens the nflammatoryi consequences of sepsis.

CRISPR-Cas9-mediated somatic correction of a one-base deletion in the Ugt1a gene ameliorates hyperbilirubinemia in Crigler-Najjar syndrome mice.

(AAV)-mediated episomal gene replacement therapy for monogenic liver disorders is currently limited in pediatric settings due to the loss of vector DNA, associated with hepatocyte duplication during liver growth. Genome editing is a promising strategy leading to a permanent and specific genome modification that is transmitted to daughter cells upon proliferation. Using genome targeting, we previously rescued neonatal lethality in mice with Crigler-Najjar syndrome. This rare monogenic disease is characterized by severe neonatal unconjugated hyperbilirubinemia, neurological damage, and death. Here, using the CRISPR-Staphylococcus aureus Cas9 (SaCas9) platform, we edited the disease-causing mutation present in the Ugt1a locus of these mice. Newborn mice were treated with two AAV8 vectors: one expressing the SaCas9 and single guide RNA, and the other carrying the Ugt1a homology regions with the corrected sequence, while maintained in a temporary phototherapy setting rescuing mortality. We observed a 50% plasma bilirubin reduction that remained stable for up to 6 months. We then tested different Cas9:donor vector ratios, with a 1:5 ratio showing the greatest efficacy in lowering plasma bilirubin, with partial lethality rescue when more severe, lethal conditions were applied. In conclusion, we reduced plasma bilirubin to safe levels and partially rescued neonatal lethality by correcting the mutant Ugt1a1 gene of a Crigler-Najjar mouse model.

Identification of 3' gene ends using transcriptional and genomic conservation across vertebrates.

BACKGROUND: In higher eukaryotes, gene expression is regulated at different levels. In particular, 3'UTRs play a central role in translation, stability and subcellular localization of transcripts. In recent years, the development of high throughput sequencing techniques has facilitated the acquisition of transcriptional data at a genome wide level. However, annotation of the 3' ends of genes is still incomplete, thus limiting the interpretation of the data generated. For example, we have previously reported two different genes, ADD2 and CPEB3, with conserved 3'UTR alternative isoforms not annotated in the current versions of Ensembl and RefSeq human databases. RESULTS: In order to evaluate the existence of other conserved 3' ends not annotated in these databases we have now used comparative genomics and transcriptomics across several vertebrate species. In general, we have observed that 3'UTR conservation is lost after the end of the mature transcript. Using this change in conservation before and after the 3' end of the mature transcripts we have shown that many conserved ends were still not annotated. In addition, we used orthologous transcripts to predict 3'UTR extensions and validated these predictions using total RNA sequencing data. Finally, we used this method to identify not annotated 3' ends in rats and dogs. As a result, we report several hundred novel 3'UTR extensions in rats and a few thousand in dogs. CONCLUSIONS: The methods presented here can efficiently facilitate the identification of not-yet-annotated conserved 3'UTR extensions. The application of these methods will increase the confidence of orthologous gene models across vertebrates.

CPEB2, CPEB3 and CPEB4 are coordinately regulated by miRNAs recognizing conserved binding sites in paralog positions of their 3'-UTRs.

The cytoplasmic polyadenylation element binding-protein (CPEB) is an RNA-binding protein that participates in translational control. CPEB2, CPEB3 and CPEB4 are paralog proteins very similar among themselves referred as the CPEB2 subfamily. To gain insight into common mechanisms of regulation of the CPEB2 subfamily transcripts, we looked for putative cis-acting elements present in the 3'-UTRs of the three paralogs. We found different families of miRNAs predicted to target all subfamily members. Most predicted target sites for these families are located in paralog positions suggesting that these putative regulatory motifs were already present in the ancestral gene. We validated target sites for miR-92 and miR-26 in the three paralogs using mutagenesis of miRNA-binding sites in reporter constructs combined with over-expression and depletion of miRNAs. Both miR-92 and miR-26 induced a decrease in Luciferase activity associated to a reduction in mRNA levels of the reporter constructs. We also showed that the endogenous miRNAs co-regulate CPEB2, CPEB3 and CPEB4 transcripts, supporting our hypothesis that these genes have a common regulatory mechanism mediated by miRNAs. We also suggest that the ancestral pattern of miRNA-binding motifs was maintained throughout the generation of highly conserved elements in each of the 3'-UTRs.

Human liver stem cells express UGT1A1 and improve phenotype of immunocompromised Crigler Najjar syndrome type I mice.

Crigler Najjar Syndrome type I (CNSI) is a rare recessive disorder caused by mutations in the Ugt1a1 gene. There is no permanent cure except for liver transplantation, and current therapies present several shortcomings. Since stem cell-based therapy offers a promising alternative for the treatment of this disorder, we evaluated the therapeutic potential of human liver stem cells (HLSC) in immune-compromised NOD SCID Gamma (NSG)/Ugt1-/- mice, which closely mimic the pathological manifestations in CNSI patients. To assess whether HLSC expressed UGT1A1, decellularised mouse liver scaffolds were repopulated with these cells. After 15 days' culture ex vivo, HLSC differentiated into hepatocyte-like cells showing UGT1A1 expression and activity. For the in vivo human cell engraftment and recovery experiments, DiI-labelled HLSC were injected into the liver of 5 days old NSG/Ugt1-/- pups which were analysed at postnatal Day 21. HLSC expressed UGT1A1 in vivo, induced a strong decrease in serum unconjugated bilirubin, thus significantly improving phenotype and survival compared to untreated controls. A striking recovery from brain damage was also observed in HLSC-injected mutant mice versus controls. Our proof-of-concept study shows that HLSC express UGT1A1 in vivo and improve the phenotype and survival of NSG/Ugt1-/- mice, and show promises for the treatment of CNSI.

Impaired motor coordination in mice lacking the EDA exon of the fibronectin gene.

The extracellular matrix (ECM) plays an important role in the central nervous system (CNS) by modulating the migration of cells, axons and dendrites of neurons. Fibronectin (FN) is a major component of the ECM in the CNS and plays essential roles in development, cell adhesion and cell migration. Specific FN-isoforms, generated by alternative splicing at three conserved regions, the extra domain B (EDB), extra domain A (EDA) and type III homologies connecting segment (IIICS), have been shown to modulate these processes in vitro and in vivo. The inclusion of the EDA exon in the brain is highly regulated during development and aging, suggesting an important role of this exon in brain function. However, the direct role of FN-isoforms in brain function and behaviour is still obscure. Therefore, to directly assess the role of the FN-EDA exon in vivo, we have generated two mouse strains devoid of EDA exon regulated splicing in the FN gene that constitutively include (EDA(+/+)) or exclude (EDA(-/-)) the EDA exon in all tissues. Here, we show the behavioural consequences of the absence of regulated splicing of the EDA exon in the FN gene. Deletion of the EDA domain in the FN protein results in reduced motor-coordination abilities and vertical exploratory capacity, whereas mice that constitutively include the EDA domain displayed a decrease in locomotory activity in the open field (OF) test. These results strongly suggest that regulated splicing of the EDA exon is necessary for a normal function of the brain.

Characterization of the distal polyadenylation site of the ß-adducin (Add2) pre-mRNA.

Most genes have multiple polyadenylation sites (PAS), which are often selected in a tissue-specific manner, altering protein products and affecting mRNA stability, subcellular localization and/or translability. Here we studied the polyadenylation mechanisms associated to the beta-adducin gene (Add2). We have previously shown that the Add2 gene has a very tight regulation of alternative polyadenylation, using proximal PAS in erythroid tissues, and a distal one in brain. Using chimeric minigenes and cell transfections we identified the core elements responsible for polyadenylation at the distal PAS. Deletion of either the hexanucleotide motif (Hm) or the downstream element (DSE) resulted in reduction of mature mRNA levels and activation of cryptic PAS, suggesting an important role for the DSE in polyadenylation of the distal Add2 PAS. Point mutation of the UG repeats present in the DSE, located immediately after the cleavage site, resulted in a reduction of processed mRNA and in the activation of the same cryptic site. RNA-EMSA showed that this region is active in forming RNA-protein complexes. Competition experiments showed that RNA lacking the DSE was not able to compete the RNA-protein complexes, supporting the hypothesis of an essential important role for the DSE. Next, using a RNA-pull down approach we identified some of the proteins bound to the DSE. Among these proteins we found PTB, TDP-43, FBP1 and FBP2, nucleolin, RNA helicase A and vigilin. All these proteins have a role in RNA metabolism, but only PTB has a reported function in polyadenylation. Additional experiments are needed to determine the precise functional role of these proteins in Add2 polyadenylation.