High frequency of TARDBP gene mutations in Italian patients with amyotrophic lateral sclerosis.

Recent studies identified rare missense mutations in amyotrophic lateral sclerosis (ALS) patients in the TARDBP gene encoding TAR DNA binding protein (TDP)-43, the major protein of the ubiquitinated inclusions (UBIs) found in affected motor neurons (MNs). The aim of this study was to further define the spectrum of TARDBP mutations in a large cohort of 666 Italian ALS patients (125 familial and 541 sporadic cases). The entire coding region was sequenced in 281 patients, while in the remaining 385 cases only exon 6 was sequenced. In 18 patients, of which six are familial, we identified 12 different heterozygous missense mutations (nine novel) all locating to exon 6, which were absent in 771 matched controls. The c.1144G>A (p.A382T) variation was observed in seven patients, thus representing the most frequent TARDBP mutation in ALS. Analysis of microsatellites surrounding the TARDBP gene indicated that p.A382T was inherited from a common ancestor in 5 of the 7 patients. Altogether, the frequency of TARDBP gene mutations appears to be particularly high in Italian ALS patients compared to individuals of mainly Northern European origin (2.7% vs. 1%). Western blot analysis of lymphocyte extracts from two patients carrying the p.A382T and p.S393L TARDBP mutations showed the presence of lower molecular weight TDP-43 bands, which were more abundant than observed in healthy controls and patients negative for TARDBP mutations. In conclusion, this report contributes to the demonstration of the causative role of the TARDBP gene in ALS pathogenesis and indicates that mutations may affect the stability of the protein even in nonneuronal tissues.

Tissue-specific splicing pattern of fibronectin messenger RNA precursor during development and aging in rat.

Fibronectin isoforms are generated by the alternative splicing of a primary transcript derived from a single gene. In rat at least three regions of the molecule are involved: EIIIA, EIIIB, and V. This study investigated the splicing patterns of these regions during development and aging, by means of ribonuclease protection analysis. Between fetal and adult rat, the extent of inclusion of the EIIIA and/or EIIIB region in fibronectin mRNA varied according to the type of tissue analyzed; but the inclusion of the V region, and in particular the V25 alternative variant, was significantly higher in all fetal than in adult tissues. These data suggest a crucial role of the V25 variant, possibly related to its interaction with the alpha 4 beta 1 integrin receptor during development. On the other hand, during aging, the only significant change observed in the splicing pattern was a decrease in the EIIIA variant in brain. The high inclusion levels of the EIIIA and EIIIB regions in young adult brain suggest that these segments may play an important role in differentiated brain tissue. The decreasing levels of inclusion of the EIIIA segment in brain fibronectin mRNA during aging may be an age-related marker with functional consequences.

Deoxyribonucleic acid polymorphism in the apolipoprotein A-1-C-III gene cluster. Association with hypertriglyceridemia.

A DNA sequence polymorphism, revealed by digestion of human DNA with the restriction endonuclease Sst-1 and hybridization with an apolipoprotein A-I complementary DNA clone, has been shown to be located in or close to the 3' noncoding region of the apolipoprotein C-III gene. This polymorphism is found in significantly increased prevalence (P less than 0.001) in Caucasian hypertriglyceridemic subjects compared with race-matched controls, and its distribution in normal individuals of differing racial origins is reported. Furthermore, no alteration of high density lipoprotein or apolipoprotein A-I and apolipoprotein C-III phenotypes was observed in individuals with or without the polymorphism.

Regulation of fibronectin EDA exon alternative splicing: possible role of RNA secondary structure for enhancer display.

The fibronectin primary transcript undergoes alternative splicing in three noncoordinated sites: the cassette-type EDA and EDB exons and the more complex IIICS region. We have shown previously that an 81-nucleotide region within the EDA exon is necessary for exon recognition and that this region contains at least two splicing-regulatory elements: a polypurinic enhancer (exonic splicing enhancer [ESE]) and a nearby silencer element (exonic splicing silencer [ESS]). Here, we have analyzed the function of both elements in different cell types. We have mapped the ESS to the nucleotide level, showing that a single base change is sufficient to abolish its function. Testing of the ESE and ESS elements in heterologous exons, individually or as part of the complete EDA regulatory region, showed that only the ESE element is active in different contexts. Functional studies coupled to secondary-structure enzymatic analysis of the EDA exon sequence variants suggest that the role of the ESS element may be exclusively to ensure the proper RNA conformation and raise the possibility that the display of the ESE element in a loop position may represent a significant feature of the exon splicing-regulatory region.

Splicing of constitutive upstream introns is essential for the recognition of intra-exonic suboptimal splice sites in the thrombopoietin gene.

The human thrombopoietin (TPO) gene, which codes for the principal cytokine involved in platelet maturation, shows a peculiar alternative splicing of its last exon, where an intra-exonic 116 nt alternative intron is spliced out in a fraction of its mRNA. To characterize the molecular mechanism underlying this alternative splicing, minigenes of TPO genomic constructs with variable exon-intron configurations or carrying exclusively the TPO cDNA were generated and transiently transfected in the Hep3B cell line. We have found that the final rate of the alternative intron splicing is determined by three elements: the presence of upstream constitutive introns, the suboptimal splice sites of the alternative intron and the length of the alternative intron itself. Our results indicate that the recognition of suboptimal intra-exonic splice junctions in the TPO gene is influenced by the assembly of the spliceosome complex on constitutive introns and by a qualitative scanning of the sequence by the transcriptional/splicing machinery complex primed by upstream splicing signals.

Influence of correct secondary and tertiary RNA folding on the binding of cellular factors to the HCV IRES.

Structural integrity of the hepatitus C virus (HCV) 5' UTR region that includes the internal ribosome entry site (IRES) element is known to be essential for efficient protein synthesis. The functional explanation for this observation has been provided by the recent evidence that binding of several cellular factors to the HCV IRES is dependent on the conservation of its secondary structure. In order to better define the relationship between IRES activity, protein binding and RNA folding of the HCV IRES, we have focused our attention on its major stem-loop region (domain III) and the binding of several cellular factors: two subunits of eukaryotic initiation factor eIF3 and ribosomal protein S9. Our results show that binding of eIF3 p170 and p116/p110 subunits is dependent on the ability of the domain III apical stem-loop region to fold in the correct secondary structure whilst secondary structure of hairpin IIId is important for the binding of S9 ribosomal protein. In addition, we show that binding of S9 ribosomal protein also depends on the disposition of domain III on the HCV 5' UTR, indicating the presence of necessary inter-domain interactions required for the binding of this protein (thus providing the first direct evidence that tertiary folding of the HCV RNA does affect protein binding).

Analysis of the linkage between fibronectin alternative spliced sites during ageing in rat tissues.

The modulation of fibronectin (FN) functions in cell-cell and cell-substrate interactions in a variety of physiological situations is achieved by the selective expression of different isoforms, which in the rat are generated by alternatively splicing of at least three regions of the molecule: EIIIA, EIIIB and V. Extensive information has been collected on the regulation of the differential processing of the single alternatively spliced regions but up to now there was scant knowledge about a possible coordinated expression of the three regions in the same transcript. Using a long range RT-PCR system we have shown that most of the splicing regulation is autonomous for each individual region but we have also observed a preferential expression of the EIIIA+ form linked to the EIIIB- variant that is age independent. Furthermore the analysis of the single regions showed interesting variations occurring in brain during the ageing. There is a decrease in the V120 form between the 6- and the 24-month-old rat brain (from 57% to 39%), whereas despite a constant prevalence of the EIIIA- form in the young rats (65%) there is a random individual variation of this form in the old animals. Noteworthy no variations have been detected in the EIIIB region (90% EIIIB-). These data suggest a role for the V and EIIIA regions, but not for the EIIIB, during the ageing process at least in brain, since no variations were detected in kidney between the 6- and 24-month-old animals.

The A/G polymorphism in the -78 position of the apolipoprotein A-I promoter does not have a direct effect on transcriptional efficiency.

A promoter polymorphism A/G at position 78 bp upstream of the transcription initiation site characterizes the human apolipoprotein A-I gene. Some studies correlated the higher Apo A-I levels or increased Apo A-I transcription efficiency with the A allele, while other studies did not confirm these results. We have investigated the in vitro effects of this transition on the transcriptional efficiency of ApoAI gene by creating two sets of identical constructs with the whole Apo A-I promoter, carrying the A or the G, linked to the complete ApoAI gene. The relative activity of the two promoter alleles was determined through a quantitative RT-PCR system after transient tranfections of human HepG2 cell line in basal state and after stimulation with retinoic acid or 17beta-estradiol. Our results exclude differences in promoter activity linked to the A or G promoter alleles either in basal or in stimulated conditions. The data suggest that the A/G polymorphism does not directly affect the transcriptional efficiency of ApoAI gene, although it may be in linkage disequilibrium with other regulatory sequences and the combination of these elements may explain the contradictory results of the ApoAI gene expression.

A family of fibronectin mRNAs in human normal and transformed cells.

Previously, two fibronectin mRNAs, generated by alternative splicing of the extra domain (ED) and type III connecting segments (IIICS) sequences, have been described in a human transformed cell line and in human liver, respectively. We now report on a family of fibronectin mRNAs identified by Northern blotting analysis in two normal human fibroblast strains (HEL 299 and Flow 7000) and five transformed cell lines (8387 and HT-1080, fibrosarcomas; G-361, melanoma; JEG-3, choriocarcinoma; and RD, rhabdomyosarcoma). Seven different fibronectin mRNA forms with electrophoretic mobilities ranging between 8.6 and 7.7 kb were identified. Each cell line contains three (HEL 299, Flow 7000 and 8387) or two (HT-1080, G-361, JEG-3 and RD) fibronectin mRNAs species with characteristic size. In all cell lines we detected one fibronectin mRNA form which lacks the ED sequence (ED- fibronectin mRNA) and one or two fibronectin mRNAs containing it (ED+ fibronectin mRNA). These data show that the presence of ED+ and ED- fibronectin mRNAs is a general feature of all cells tested. Moreover, the fibronectin mRNA pattern is characteristic of the cell type analyzed, suggesting the occurrence of specifically programmed splicing mechanisms in each cell line.

Mammalian cell expression of malaria merozoite surface proteins and experimental DNA and RNA immunisation.

The gene for a 45 kDa merozoite surface protein (MSA-2) of the human malaria parasite Plasmodium falciparum was PCR amplified and cloned into eukaryotic expression vectors VR1012 and pcDNA3 to yield plasmids P1 and P2, respectively. The coding sequences for two N-terminal fragments of the 185 kDa merozoite surface protein (MSA-1) gene were similarly PCR amplified and cloned into vectors VR1020 and VR1012 to yield plasmids P3 and P4, respectively. The MSA-1 signal peptide sequence, present in P4, was replaced with the human tissue plasminogen activator signal sequence in P3. The four plasmids expressed the cloned genes under the control of the cytomegalovirus promoter and carried 3' bovine growth hormone termination/poly A signals. P1, P3 and P4 also contained the cytomegalovirus intron A enhancer sequence. MSA-1 expression was more readily detected than MSA-2 in Cos cells transfected with P3/P4 and P1/P2 respectively. The MSA-2 gene was also cloned into the phagemid pBluescript IISK+ with and without a 3' poly A tail composed of 35 A residues. MSA-2 was synthesised in HeLa cells infected with a recombinant vaccinia virus carrying T7 RNA polymerase when MSA-2 recombinant pBluescript was transfected into the cells. Inoculation with P1 intramuscularly or intradermally and with P2 intradermally into rabbits led to the production of antibodies to MSA-2 detectable by immunofluorescence and Western blotting. Antibodies were also produced against MSA-1 after intramuscular/intradermal inoculation with P3 and P4. Inoculation of rabbits with MSA-2 mRNA yielded better antibody titres when a poly A tail was present. Antibody levels were maintained for > 9 weeks after the final immunisation. However the immune sera failed to inhibit in vitro parasite growth.

Comparison of the human apolipoprotein genes. Apo AII presents a unique functional intron-exon junction.

The structure and function of the apolipoproteins are of interest because of their central role in lipid metabolism. We report the complete primary structure of the human apo AII and CIII genes. These, like apo AI, contain three introns located at conserved positions. This may reflect their evolutionary and functional relationship. Indeed, computer-aided analysis shows that these apolipoproteins and apo AIV (rat), CI, CII and E contain homologous amino acid sequences. In the case of apo AI, AII and CIII, such sequences are encoded by equivalent exons. Finally, the apo AII gene presents a unique intron-exon junction sequence 5' (G-T)16G-G-G-C-A-G 3' that, although departing considerably from the accepted consensus (Py)15X-Py-A-G, appears to function efficiently both in vivo and in a transient expression system.

An age-related reduction of brain TBPH/TDP-43 levels precedes the onset of locomotion defects in a Drosophila ALS model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. The average age of onset of both sporadic and familial cases is 50-60 years of age. The presence of cytoplasmic inclusions of the RNA-binding protein TAR DNA-binding protein-43 (TDP-43) in the affected neurons is seen in 95% of the ALS cases, which results in TDP-43 nuclear clearance and loss of function. The Drosophila melanogaster ortholog of TDP-43 (TBPH) shares many characteristics with the human protein. Using a TDP-43 aggregation inducer previously developed in human cells, we created a transgenic fly that shows an adult locomotive defect. Phenotype onset correlates with a physiologically age-related drop of TDP-43/TBPH mRNA and protein levels, seen both in mice and flies. Artificial reduction of mRNA levels, in vivo, anticipates the locomotion defect to the larval stage. Our study links, for the first time, aggregation and the age-related, evolutionary conserved reduction of TDP-43/TBPH levels with the onset of an ALS-like locomotion defect in a Drosophila model. A similar process might trigger the human disease.

Characterisation and chromosomal localisation of the rat alpha- and beta-adducin-encoding genes.

A polymorphism in the genes encoding alpha- and beta-adducin (ADD) was described as being associated with blood-pressure variation in a genetically hypertensive strain of rats (MHS). ADD is a cytoskeletal heterodimeric protein which may be involved in cellular signal transduction and interacts with other membrane skeleton proteins which affect ion transport across the cell membrane. The cDNA encoding the alpha subunit of rat ADD was isolated using PCR methods. The cDNA consists of about 3900 bp and encodes a protein of 735 amino acids (aa) which shows 91% aa identity with the human counterpart. In spleen and kidney, three alternative spliced exons were found by PCR amplification and confirmed by RNase protection analysis. 17 inbred rat strains were genotyped for the polymorphism in the alpha- and beta-ADD genes. Chromosomal localisation mapped rat alpha-ADD on chromosome 14 and rat beta-ADD on chromosome 4.

Genomic organisation and chromosomal localisation of the gene encoding human beta adducin.

Adducin (ADD) is a heterodimeric protein of the membrane skeleton with subunits of 103 (alpha) and 97 kDa (beta). It promotes the assembly of the spectrin-actin network. We have previously shown that one point mutation in each of the alpha and beta rat ADD-encoding genes is associated with blood pressure variation in an animal model for hypertension, the Milan hypertensive strain of rats, probably due to a change in the phosphorylation pattern. In fact, the rat mutations, Y to F for alpha and R to Q for beta, are located, respectively, in a tyrosine kinase and a protein kinase A phosphorylation site. We have now determined, for the human beta-ADD-encoding gene, its chromosomal localisation, exon-intron organisation and alternative splicing patterns. We report here that human beta-ADD is localised on chromosome 2 and we also show a characteristic 3' end alternative splicing of the beta-ADD RNA that generates two distinct beta-ADD families, namely ADD 63 and 97; both of them in turn present a very complex differential splicing pattern in the internal exons.

Exon structure of the collagen-binding domain of human fibronectin.

Fibronectin consists of a series of three internal homology repeats (types I, II, and III [(1983) Proc. Natl. Acad. Sci. USA 80, 137-141]). The type III units are each coded for by two exons with the exception of the alternatively spliced extra domain type III [(1984) EMBO J. 3, 2511-2516]. To investigate the gene organisation of the type I and II repeats, genomic clones covering the collagen-binding domain of human fibronectin have been isolated and characterised. The results show that each of the type I and II homology units in this region corresponds to an exon. Taken together with the previous data concerning the type III units, the data lend support to a gene fusion model of fibronectin evolution.

Isolation and characterization of cDNA clones for human liver fibronectin.

Cellular and plasma fibronectin dimers are constituted by similar but not identical polypeptides. Their differences are the consequence of internal primary sequence variability due to alternative splicing in at least 2 regions (ED and IIICS) of the pre mRNAs [1-8]. A detailed analysis of human liver fibronectin mRNA in these regions was carried out by isolating cDNA clones and determining their nucleotide sequence. A novel type of IIICS segment (coding for 64 amino acids) was present in the two cDNA clones studied and, as expected from previous S1 mapping studies [6], the ED segment was absent in both.

Donor and acceptor splice signals within an exon of the human fibronectin gene: a new type of differential splicing.

We have sequenced that area of a human fibronectin gene clone which codes for a connecting strand separating the last two areas of the type III homology. The gene has a complex exon with two 'AG' acceptor sites. One of these can be used (exon subdivision). In addition 93 basepairs inside the exon are sometimes spliced out as an intron. This is the third differential splicing found in the fibronectin gene transcript and it represents a new type of differential splicing.

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.

L273S missense substitution in human lysosomal acid lipase creates a new N-glycosylation site.

Human lysosomal acid lipase (LAL), when expressed in HeLa cells using the Vaccinia T7 expression system, showed four major molecular forms ranging from 42 to 54 kDa. Treatment with endoglycosidase H resulted in a 42 kDa protein, indicating that the molecular weight variations were due to N-glycosylation. A missense substitution, L273S, previously detected in a patient with cholesteryl ester storage disease (CESD), produced catalytically inactive LAL showing a largest molecular mass form of 56 kDa instead of 54 kDa. Analysis of the amino acid sequence in the close proximity of the mutation (NMS- NML) indicated that the L273S mutation creates an additional N-glycosylation consensus (N-X-S/T) in this region. Two site directed mutants disrupting this consensus, QMS and QML, when expressed in HeLa cells, did not show the 56 kDa form but the normal 54 kDa band whereas deglycosylation always resulted in the major 42 kDa form, as observed with normal LAL and the L273S mutant. These data confirmed that an additional N-glycosylation at N271 was responsible for the 56 kDa form of the protein produced from the L273S allele. Furthermore, deglycosylation of normal LAL reduced the acid hydrolase activity towards both tri-oleyl glycerol and cholesteryl oleate by 50%, strongly suggesting that N-linked carbohydrate residues are important for optimal catalytic activity.

Transcription efficiency of human apolipoprotein A-I promoter varies with naturally occurring A to G transition.

In human, the gene coding for apolipoprotein A-I (apo A-I), a protein of the plasma lipid transport system, is expressed only in the liver and the intestine. A naturally occurring A to G substitution in the promoter at position -78 was shown to be associated with high density lipoproteins (HDL) in females. We have studied the effect of this mutation on promoter activity using various lengths of promoter sequences and the CAT reporter gene system. Transient expression studies after introduction of these constructs into Hep 3B cells revealed that in the region spanning -330 to +1 of the promoter an A to G substitution increases the activity approximately twofold. On the other hand, when further upstream region (-1469 to +397) is also included, the promoter activity seems comparable in both alleles. Our results show how minimal sequence variations can affect the in vitro analysis of promoter activity.