Alessandra Iaconcig

Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan

Fibronectins (FNs) are multifunctional high molecular weight glycoproteins present in the blood plasma and in the ECMs of tissues. The FN primary transcript undergoes alternative splicing in three regions generating up to 20 main different variants in humans. However, the precise role of the FN isoforms is poorly understood. One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging. To study its in vivo function, we generated mice devoid of EDA exon-regulated splicing. Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP–mediated deletion of the exon. Homozygous mouse strains with complete exclusion or inclusion of the EDA exon were viable and developed normally, indicating that the alternative splicing at the EDA exon is not necessary during embryonic development. Conversely, mice without the EDA exon in the FN protein displayed abnormal skin wound healing, whereas mice having constitutive inclusion of the EDA exon showed a major decrease in the FN levels in all tissues. Moreover, both mutant mouse strains have a significantly shorter lifespan than the control mice, suggesting that EDA splicing regulation is necessary for efficient long-term maintenance of biological functions.

RNA folding affects the recruitment of SR proteins by mouse and human polypurinic enhancer elements in the fibronectin EDA exon.

ABSTRACT In humans, inclusion or exclusion of the fibronectin EDA exon is mainly regulated by a polypurinic enhancer element (exonic splicing enhancer [ESE]) and a nearby silencer element (exonic splicing silencer [ESS]). While human and mouse ESEs behave identically, mutations introduced into the homologous mouse ESS sequence result either in no change in splicing efficiency or in complete exclusion of the exon. Here, we show that this apparently contradictory behavior cannot be simply accounted for by a localized sequence variation between the two species. Rather, the nucleotide differences as a whole determine several changes in the respective RNA secondary structures. By comparing how the two different structures respond to homologous deletions in their putative ESS sequences, we show that changes in splicing behavior can be accounted for by a differential ESE display in the two RNAs. This is confirmed by RNA-protein interaction analysis of levels of SR protein binding to each exon. The immunoprecipitation patterns show the presence of complex multi-SR protein-RNA interactions that are lost with secondary-structure variations after the introduction of ESE and ESS variations. Taken together, our results demonstrate that the sequence context, in addition to the primary sequence identity, can heavily contribute to the making of functional units capable of influencing pre-mRNA splicing.

A Major Fraction of Fibronectin Present in the Extracellular Matrix of Tissues Is Plasma-derived

The origin of the fibronectin (FN) found in the extracellular matrix of tissues has not been defined experimentally. Previous studies suggest that there is contribution from both local tissue production and transfer from plasma, but the extent of this phenomenon has not been addressed. We have shown before that engineered mice constitutively expressing extra domain A-containing FN (EDA+FN) have a significant decrease of FN levels in plasma and most tissues. We showed that hepatocytes modified to produce EDA+FN have normal extracellular matrix-FN levels but secrete less soluble FN. When we performed a liver-specific EDA-exon deletion in these animals, FN levels were restored both in plasma and tissues. Therefore, an important fraction of tissue FN, approximately an equal amount of that produced by the tissue itself, is actually plasma-derived, suggesting that plasma is an important source of tissue FN. The present results have potential significance for understanding the contributions of plasma FN, and perhaps other plasma proteins, in the modulation of cellular activities and in the formation of the extracellular matrix of tissues.

Regulation of the fibronectin EDA exon alternative splicing. Cooperative role of the exonic enhancer element and the 5′ splicing site

Alternatively spliced exons generally contain weak splicing sites, and exonic and/or intronic regulatory elements recognised by trans‐acting auxiliary splicing factors. The EDA exon of the fibronectin gene is a typical example of an exon bearing a purine‐rich exon splicing enhancer (ESE) element recognised by members of the SR phosphoprotein family. The regulatory region that governs splicing in the human EDA exon also contains an exon splicing silencer (ESS) element. We have cloned the mouse EDA genomic region, and we show that the ESE and the ESS elements, although they have base differences, can be replaced by the human elements without significant change in the exon inclusion/exclusion ratio. This fact suggests a common splicing regulatory mechanism across species. We demonstrate in vivo the functional activity of the mouse ESE element in splicing. We also show that the trans‐acting factors recognising this element cooperate with the 5′ splicing site of the EDA exon to facilitate proper exon recognition. Indeed, a strong 5′ splicing site overrides the ESE function in exon recognition. However, the presence of a strong 3′ splicing site is not sufficient to compensate for the absence of the splicing enhancer. Our data provide in vivo evidence of the interplay between the exonic splicing regulatory elements and the splicing sites, leading finally to subtle regulation of alternative splicing.

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.

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.

TDP-43 regulates β-adducin (Add2) transcript stability

TDP-43 is an RNA-binding protein involved in several steps of mRNA metabolism including transcription, splicing and stability. It is also involved in ALS and FTD, neurodegenerative diseases characterized by TDP-43 nuclear depletion. We previously identified TDP-43 as a binder of the downstream element (DSE) of the β-Adducin (Add2) brain-specific polyadenylation site (A4 PAS), suggesting its involvement in pre-mRNA 3′ end processing. Here, by using chimeric minigenes, we showed that TDP-43 depletion in HeLa and HEK293 cells resulted in down-regulation of both the chimeric and endogenous Add2 transcripts. Despite having confirmed TDP-43-DSE in vitro interaction, we demonstrated that the in vivo effect was not mediated by the TDP-43-DSE interaction. In fact, substitution of the Add2 DSE with viral E-SV40 and L-SV40 DSEs, which are not TDP-43 targets, still resulted in decreased Add2 mRNA levels after TDP-43 downregulation. In addition, we failed to show interaction between TDP-43 and key polyadenylation factors, such as CstF-64 and CPSF160 and excluded TDP-43 involvement in pre-mRNA cleavage and regulation of polyA tail length. These evidences allowed us to exclude the pre-hypothesized role of TDP43 in modulating 3′ end processing of Add2 pre-mRNA. Finally, we showed that TDP-43 regulates Add2 gene expression levels by increasing Add2 mRNA stability. Considering that Add2 in brain participates in synapse assembly, synaptic plasticity and their stability, and its genetic inactivation in mice leads to LTP, LTD, learning and motor-coordination deficits, we hypothesize that a possible loss of Add2 function by TDP-43 depletion may contribute to ALS and FTD disease states.

Unexpected rescue of alpha-synuclein and multimerin1 deletion in C57BL/6JOlaHsd mice by beta-adducin knockout

Uniform genetic background of inbred mouse strains is essential in experiments with genetically modified mice. In order to assess Add2 (beta-adducin) function, its null mutation was produced in embryonic stem cells derived from 129Sv mouse and the subsequently obtained mouse mutants were backcrossed for 6 generations with C57BL/6JOlaHsd strain. Comparison of brain proteins between mutated and control animals by two-dimensional gels linked to mass spectroscopy analysis showed expression of Snca (alpha-synuclein) in the mutated animals, but unexpectedly not in the control C57BL/6JOlaHsd mice. Comparison between C57BL/6JOlaHsd and C57BL/6NCrl mice confirmed the presence of a deletion encompassing Snca and in addition Mmrn1 (multimerin1) loci in C57BL/6JOlaHsd strain. The segregation of mutated Add2 together with an adjacent part of the chromosome 6 derived from 129Sv mice, rescued the loss of these two genes in knockout mice on C57BL/6JOlaHsd background. The fact that Add2 knockout was compared with the C57BL/6JOlaHsd mouse strain, which is actually a double knockout of Snca and Mmrn1 emphasizes a need for information provided by commercial suppliers and of exact denominations of substrains used in research.

Modulation of bilirubin neurotoxicity by the Abcb1 transporter in the Ugt1-/- lethal mouse model of neonatal hyperbilirubinemia.

Moderate neonatal jaundice is the most common clinical condition during newborn life. However, a combination of factors may result in acute hyperbilirubinemia, placing infants at risk of developing bilirubin encephalopathy and death by kernicterus. While most risk factors are known, the mechanisms acting to reduce susceptibility to bilirubin neurotoxicity remain unclear. The presence of modifier genes modulating the risk of developing bilirubin-induced brain damage is increasingly being recognised. The Abcb1 and Abcc1 members of the ABC family of transporters have been suggested to have an active role in exporting unconjugated bilirubin from the central nervous system into plasma. However, their role in reducing the risk of developing neurological damage and death during neonatal development is still unknown.To this end, we mated Abcb1a/b-/- and Abcc1-/- strains with Ugt1-/- mice, which develop severe neonatal hyperbilirubinemia. While about 60% of Ugt1-/- mice survived after temporary phototherapy, all Abcb1a/b-/-/Ugt1-/- mice died before postnatal day 21, showing higher cerebellar levels of unconjugated bilirubin. Interestingly, Abcc1 role appeared to be less important.In the cerebellum of Ugt1-/- mice, hyperbilirubinemia induced the expression of Car and Pxr nuclear receptors, known regulators of genes involved in the genotoxic response.We demonstrated a critical role of Abcb1 in protecting the cerebellum from bilirubin toxicity during neonatal development, the most clinically relevant phase for human babies, providing further understanding of the mechanisms regulating bilirubin neurotoxicity in vivo. Pharmacological treatments aimed to increase Abcb1 and Abcc1 expression, could represent a therapeutic option to reduce the risk of bilirubin neurotoxicity.

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.