New papers from Professor Baralle's lab

Marcucci, R., Romano, M., Feiguin, F., O'Connell, M., Baralle, F.E. Dissecting the splicing mechanism of the Drosophila editing enzyme; dADAR. 2009. Nucleic Acids Res. 37(5), 1663-1671

Abstract: In Drosophila melanogaster, the expression of adenosine deaminase acting on RNA is regulated by transcription and alternative splicing so that at least four different isoforms are generated that have a tissue-specific splicing pattern. Even though dAdar has been extensively studied, the complete adult expression pattern has yet to be elucidated. In the present study, we investigate mature transcripts of dAdar arising from different promoters. Two predominant isoforms of dAdar are expressed in gonads and dAdar is transcribed from both the embryonic and the adult promoters.
Furthermore, full-length transcripts containing the alternatively spliced exon-1 are expressed in a tissue-specific manner. The splicing factor B52/SRp55 binds within the alternative spliced exon 3a and plays a role in this alternative splicing event.

Zago, P., Baralle, M., Ayala, Y.M., Skoko, N., Zacchigna, S., Buratti, E., Tisminetzky, S. Improving human beta interferon production in mammalian cell lines by insertion of an intronic sequence within its naturally uninterrupted gene. 2009. Biotechnol. Appl. Biochem. 52(3), 191-198

Abstract: Human beta-interferon is used extensively as a therapeutic agent in a wide variety of diseases, ranging from multiple sclerosis to viral infections. At present, the most common source of interferon-beta is derived from CHO (Chinese-hamster ovary) cells. Interestingly, however, the IFNB gene is characterized by a lack of intronic sequences and therefore does not undergo splicing during its expression pathway. As nuclear processing of pre-mRNA molecules has often been demonstrated to improve production yields of recombinant molecules, we have inserted a heterologous intronic sequence at different positions within the IFNB gene and analysed its effects on protein production. The results obtained in the present study show that the position of intron insertion has profound effects on the expression levels of the IFNB gene and on the nuclear/cytoplasm distribution levels of its mRNA as determined by FISH (fluorescent in situ hybridization) analysis of stably transfected clones. In conclusion, our results provide additional evidence that insertion of intronic sequences may be used to improve protein expression efficiency also in molecules that do not normally undergo any splicing process.

Raponi, M., Buratti, E., Dassie, E., Upadhyaya, M., Baralle, D. Low U1 snRNP dependence at the NF1 exon 29 donor splice site. 2009. FEBS J. 276(7), 2060-2073

doi: 10.1111/j.1742-4658.2009.06941.x

Abstract: Many disease-causing splicing mutations described in the literature produce changes in splice sites (SS) or in exon-regulatory sequences. The delineation of these splice aberrations can provide important insights into novel regulation mechanisms. In this study, we evaluated the effect of patient variations in neurofibromatosis type 1 (NF1) exon 29 and its 5'SS surrounding area on its splicing process. Only two of all nonsense, missense, synonymous and intronic variations analyzed in this study clearly altered exon 29 inclusion/exclusion levels. In particular, the intronic mutation +5g>a had the strongest effect, resulting in total exon exclusion. This finding prompted us to evaluate the exon 29 5'SS in relation to its ability to bind U1 snRNP. This was performed by direct analysis of the ability of U1 to bind to wild-type and mutant donor sites, by engineering an in vitro splicing system to directly evaluate the functional importance of U1 snRNA base pairing with the exon 29 donor site, and by coexpression of mutant U1 snRNP molecules to try to rescue exon 29 inclusion in vivo. The results revealed a low dependency on the presence of U1 snRNP, and suggest that exon 29 donor site definition may depend on alternative mechanisms of 5'SS recognition.

Corrado, L., Ratti, A., Gellera, C., Buratti, E., Castellotti, B., Carlomagno, Y., Ticozzi, N., Mazzini, L., Testa, L., Taroni, F., Baralle, F.E., Silani, V., D'Alfonso, S. High frequency of TARDBP gene mutations in Italian patients with Amyotrophic Lateral Sclerosis. 2009. Hum. Mutat. 30(4), 688-694

doi: 10.1002/humu.20950

Abstract: 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.