Direzione e uffici C.I.B.

Direzione CIB:
Prof. Claudio Schneider
Email: claudio.schneider@Lncib.it

Segreteria CIB:
Prof. Roberto Gambari
Email: roberto.gambari@unife.it

SEGRETERIA ORGANIZZATIVA:
Elisabetta Lambertini
Tel: 0532/974451
Fax: 0532/974484
E-mail: lmblbt@unife.it

AMMINISTRAZIONE:
Vanessa Florit
Area di Ricerca
Padriciano, 99 - 34012 Trieste
Tel: 040/398979
Fax: 040/398990
E-mail: cib@lncib.it

Posta certificata C.I.B.:
cib@poste-certificate.it

Login



Pietropaolo Concetta Stampa
RESPONSABILE DELLA U. O.

Cognome e Nome
Pietropaolo Concetta
Qualifica
Professore Ordinario
Facoltà Farmacia
Dipartimento Biochimica e Biotecnologie Mediche
Settore Scientifico Disciplinare BIO/10
E-mail Questo indirizzo e-mail è protetto dallo spam bot. Abilita Javascript per vederlo.

PERSONALE STRUTTURATO

Cognome e Nome ARCONE ROSARIA
Qualifica Professore Associato
Dipartimento Scienze Farmaco-biologiche
Ente di appartenenza
Università di Napoli Federico II
Cognome e Nome
PIETROPAOLO CONCETTA
Qualifica
Professore Ordinario
Dipartimento Biochimica e Biotecnologie Mediche
Ente di appartenenza
Università di Napoli Federico II
Cognome e Nome
RUSSO GIULIA
Qualifica Professore Associato
Dipartimento Biochimica e Biotecnologie Mediche
Ente di appartenenza
Università di Napoli Federico II
Cognome e Nome
Qualifica
Dipartimento
Ente di appartenenza
Cognome e Nome
Qualifica
Dipartimento
Ente di appartenenza
Cognome e Nome
Qualifica
Dipartimento
Ente di appartenenza

PERSONALE NON STRUTTURATO

Cognome e Nome RUSSO ANNAPINA
Qualifica Assegnista di ricerca
Dipartimento Biochimica e Biotecnologie Mediche
Ente di appartenenza
Università di Napoli Federico II
Cognome e Nome SICILIANO GABRIELLA
Qualifica
Dottoranda
Dipartimento Biochimica e Biotecnologie Mediche
Ente di appartenenza
Università di Napoli Federico II
Cognome e Nome
Qualifica
Dipartimento
Ente di appartenenza
Cognome e Nome
Qualifica
Dipartimento
Ente di appartenenza
Cognome e Nome
Qualifica
Dipartimento
Ente di appartenenza
Cognome e Nome
Qualifica
Dipartimento
Ente di appartenenza

LINEE DI RICERCA

Research project n.1 Title:Nonsense-mediated decay (NMD) and spatial distribution of mammalian ribosomal protein mRNAs in the post-transcriptional regulation of mammalian ribosomal protein genes Gene expression is mediated by RNA molecules, whose accurate biogenesis and distribution are mostly responsible for the accuracy of gene products. Eucaryotic cells have developed during evolution a variety of mechanisms to exert a quality control of mRNAs at various levels of post-transcriptional and translational processes. Eventually, the amounts and the quality of gene products are a result of an efficient regulation of the surveillance processes. Nonsense-mediated mRNA decay (NMD) is a process of rapid and selective degradation of mRNAs containing a premature stop-codon (PTC), as a result of a mutation in DNA of germ or somatic cells, or as result of an inefficient splicing during the post-transcriptional processing. The resulting mRNA is substrate of NMD, which couples down-regulation of translation and activation of the degradation pathway of mRNA itself. Identification and characterization of molecular mechanisms mediating this mRNA quality control process are the major aim of studies in several laboratories. The NMD process initiates in the nucleus when a set of proteins collectively called the exon-junction complex (EJC) is deposited 20-24 nucleotides upstream from each exon-exon junction after RNA splicing. The first translating ribosome, in a so-called pioneer round of translation, will displace the EJC from all exon-exon junctions, but the EJC positioned downstream from PTC. Genetic studies in C. elegans have led to the identification of seven genes (smg 1-7) involved in NMD in higher eukaryotes. Some smg genes have been cloned and gene products characterized. NMD has been mostly studied as a control mechanism preventing the production of truncated dominant negative polypeptides, potentially deleterious to the organism. However, many data indicate that NMD can be involved in the regulation of the expression of a number of wildtype genes, by regulating the half-life of mRNA isoforms generated by alternative splicing. Support for a physiological role of NMD comes from the analysis of the human EST database, which led to the prediction that up to 35% of splicing events in human gene transcripts lead to PTC-containing mRNAs, which are putative substrates for NMD. Among others, 11 ribosomal protein genes have been predicted to generate aberrant mRNAs. The r-proteins are present in equimolar amounts in the ribosome, and their production is strictly regulated at various gene expression levels. In eukaryotes, the synthesis of ribosomal proteins is controlled mostly via both post-transcriptional and translational control mechanisms. In fact, it has been demonstrated that the growth-dependent translation of r-protein mRNA (rp-mRNA) is mediated by their shift from polysomes in growing cells, to mRNP particles in quiescent cells. A selective translational repression of rp-mRNAs has been observed during differentiation and development in Xenopus and Drosophila. Post-transcriptional control, in some cases, is obtained through feedback regulation mechanisms similar to those found in E. coli. The Xenopus laevis rpL4 is feedback-regulated via splicing of precursor mRNA and the human homologue of rpS14 is feedback-regulated at transcription level. In fact, a finely modulated production of r-proteins is essential to balance a correct ribosome biogenesis and their extraribosomal functions. In the past few years we have studied human ribosomal protein genes, transport of r-proteins in the nucleus and the nucleolar accumulation, as well as RNA-rprotein interactions; we are currently studying the relevance of NMD in the post-transcriptional regulation of mammalian rp-genes. We have identified alternatively spliced mRNA transcripts of genes encoding rpL3 and rpL12, and we have showed that they are natural targets of NMD. We also demonstrated that the production of the rpL3 NMD-sensitive transcript strictly depends on rpL3 abundance. In fact, over-expression of rpL3 in rat PC12 cells alters the splicing pattern so that the NMD-target isoform increases and the canonically spliced rpL3 mRNA decreases. These results indicate that rpL3 auto-regulates its expression by coupling alternative splicing and NMD. However, the molecular mechanisms through which a r-protein can regulate the splicing of its own gene are not yet understood; our research will aim to contribute data to understand the relevance and the mechanisms of the transcriptional regulation, via NMD, of r-protein genes. Many data support the notion that the intracellular distribution of transcripts plays a critical role in cell organization and development in organisms such as Drosophila or Xenopus. Although observed primarily in highly polarized cells, an asymmetrical distribution of mRNAs has been observed also in other mammalian cells. For example, in fibroblasts the mRNA encoding the nuclear protein c-myc localizes at the perinuclear cytoplasm, whereas OXA1 mRNA localizes in the proximity of mitochondria. According to the most plausible hypothesis, this subcellular localization results in efficient production of encoded proteins in the cell region where they are required, and eventually facilitates their efficient targeting. The molecular mechanisms involved in mRNA localization are not fully determined. Although a combination of mechanisms may be used to localize mRNAs, in most systems correct localization of transcripts depends on the integrity of the cytoskeleton, because it occurs by way of a microtubule-dependent mechanism, or by way of actin microfilaments. Also in non-polarized mammalian cells, transcripts such as c-myc are associated with the cytoskeleton. Thus, interaction of mRNA with the cytoskeletal network appears to be a general mechanism of asymmetric accumulation of certain mRNAs at specific cytoplasmic sites. All localized mRNAs contain cis-acting elements that are essential for their subcellular localization and most localized mRNAs contain targeting sequences, termed zip-codes, in their 3’-UTR. Various attempts have been made also to identify the proteins involved, in trans, in the diverse steps of the localization process. Cytosolic and mitochondrial ribosomal proteins (r-proteins) undergo intense cell trafficking. In fact, they are encoded by the nuclear genome and are eventually localized in distinct, specific cytoplasmic regions. Cytosolic r-proteins, after being synthesized in the cytoplasm, are transported into the nucleus to assemble, in the nucleolus, with nascent rRNA. Thus, rapid transfer into the nucleus is crucial because unassembled r-proteins are toxic to the cell and are rapidly degraded. It has been reported that mRNAs encoding cytosolic rpL4 and rpS6 are mostly associated with cytoskeleton-bound polysomes. These findings led to postulate that by binding to the cytoskeleton, mRNAs coding for nuclear proteins would be retained in the perinuclear cytoplasm and so promote efficient transport of the newly synthesized proteins to the nucleus. Mitochondrial biogenesis, on the other hand, needs the expression of both nuclear and mitochondrial genomes, and mitochondrial r-proteins must be correctly addressed to the mitochondrial compartment in synchrony with biogenesis. Thus, it is conceivable that also the transcripts encoding mitochondrial r-proteins are asymmetrically distributed in the cytoplasm. Support for this hypothesis comes from the identification in yeast of >100 mRNAs that encode mitochondrial proteins associated to mitochondria-bound polysomes. In the light of these data, our studies are aimed to determine the subcellular distribution of mRNAs that encode human cytosolic and mitochondrial r-proteins, and to identify the transcript regions containing the signal that directs the mRNA to a specific cytoplasmic region.

Research project n.2 Title: Serpin protease nexin 1 (PN1) in neuronal and glial cell differentiation PN1 is a member of the serpin superfamily which mainly inhibits thrombin; the inhibition is enhanced by the interaction with heparin. PN1 has been implicated in embryogenesis of the nervous system and of sexual organs and probably plays a role in a variety of pathologies including Alzheimer disease and scleroderma. In addition, studies in cell colture have suggested that the balance thrombin-PN1 might contribute to the nervous system plasticity, during development as well as in healing processes following tissue damage. To better understand PN1 functions, we have designed experimental cell systems to achieve an inducible over-expression of PN1, or silencing of its gene. To achieve an inducible overexpression of PN1, the tet-off system has been utilized in PC12 cell line, rat pheocromocitoma cells undergoing neuronal diffentiation when exposed to Nerve Growth Factor (NGF). The PC12-Tet-off cells have been engineered to express PN1 under control of doxycyclin. Stably transfected clones were isolated and analyzed for inducible PN1 expression, by western blot on protein lysates, and by semi-quantitative RT-PCR on RNA. The clones showing the highest induction of PN1 expression were characterized and the morphological differentiation evaluated following exposure to NGF. This cellular system represents a valuable tool to study the modulation of neuronal differentiation by NGF as well as by interactions thrombin-PN1. To achieve silencing of PN1 gene, we have designed a RNA interference protocol to silence PN1 gene in C6 cells, derived from rat glioma cells and constitutively producing high levels of PN1. To this aim, oligonucleotides, specifically designed to activate the RNA interfering process, were cloned and expressed under control of U6 snRNA promoter. The analysis of RNA, by Northern blot, showed a variable silencing of PN1 depending on the transfected construct, the highest silencing reaching 60%. The analysis of protein, by Western blot, showed 80% protein silencing insome clones. Stably transfected clones have been isolated and are available to perform studies on glial differentiation of C6 cells and role of PN1 in glial cell differentiation.

TECNOLOGIE IN POSSESSO DELL'U. O.

  • In situ hybridization
  • Quantitative Real Time PCR
  • Live cell time-lapse microscopy

STRUMENTAZIONE

Denominazione
Struttura ove la strumentazione è allocata
Responsabile della strumentazione
Molecular Imager FX ProPlus Multimager system (Biorad)
Laboratori DBBM, Edificio 18
Annapina Russo
ABI PRISM 3100 Genetic analyzer
Laboratori DBBM, Edificio 18
Giulia Russo
In vivo cell imaging system: Zeiss Axiovert 200 microscope, telecamera, computer and accessories required for time-lapse microscopy
Laboratori DBBM, Edificio 18
Annapina Russo

PUBBLICAZIONI

1. Cuccurese M, Russo G, Russo A, Pietropaolo C. Alternative splicing and nonsense-mediated mRNA decay regulate mammalian ribosomal gene expression. Nucleic Acids Res. 33:5965-5977, 2005.
2. Russo A, Russo G, Peticca M, Pietropaolo C, Di Rosa M, Iuvone T. Inhibition of granuloma-associated angiogenesis by controlling mast cell mediator release: role of mast cell protease-5. Br J Pharmacol. 145:24-33,2005
3. Russo G, Cuccurese M, Monti G, Russo A, Amoresano A, Pucci P, Pietropaolo C. Ribosomal protein L7a binds RNA through two distinct RNA-binding domains. Biochem J. 385(Pt 1):289-299, 2005
3. Russo G, Cuccurese M, Monti G, Russo A, Amoresano A, Pucci P, Pietropaolo C. Ribosomal protein L7a binds RNA through two distinct RNA-binding domains. Biochem J. 385(Pt 1):289-299, 2005
4. Orcutt SJ, Pietropaolo C, Krishnaswamy S. Extended interactions with prothrombinase enforce affinity and specificity for its macromolecular substrate. J Biol Chem. 277:46191-46196, 2002
5. Blasi F, Ciarrocchi A, Luddi A, Strazza M, Riccio M, Santi S, Arcone R, Pietropaolo C, D'Angelo R, Costantino-Ceccarini E, Melli M. Stage-specific gene expression in early differentiating oligodendrocytes. Glia. 39:114-123, 2002
6. Angiolillo A, Russo G, Porcellini A, Smaldone S, D'Alessandro F, Pietropaolo C. The human homologue of the mouse Surf5 gene encodes multiple alternatively spliced transcripts. Gene. 284:169-178,2002
7. Festa M, Ricciardelli G, Mele G, Pietropaolo C, Ruffo A, Colonna A. Overexpression of H ferritin and up-regulation of iron regulatory protein genes during differentiation of 3T3-L1 pre-adipocytes. J Biol Chem. 275:36708-36712, 2000
8. Loew D, Perrault C, Morales M, Moog S, Ravanat C, Schuhler S, Arcone R, Pietropaolo C, Cazenave JP, van Dorsselaer A, Lanza F. Proteolysis of the exodomain of recombinant protease-activated receptors: prediction of receptor activation or inactivation by MALDI mass spectrometry. Biochemistry.39:10812-10822,2000
9. Festa M, Colonna A, Pietropaolo C, Ruffo A. Oxalomalate, a competitive inhibitor of aconitase, modulates the RNA-binding activity of iron-regulatory proteins. Biochem J. 348 Pt 2:315-320, 2000

DOTTORATI DI RICERCA

Componente U.O. Dottorato di Ricerca Coordinatore Sede
Pietropaolo Concetta
Biochimica e Biologia cellulare e molecolare
Giuseppe D’Alessio
Dipartimento di Biologia strutturale e funzionale
Russo Giulia
Biochimica e Biologia cellulare e molecolare
Giuseppe D’Alessio
Dipartimento di Biologia strutturale e funzionale

CONGRESSI C.I.B.

Congressi Partecipazione
CNB4

CNB5

CNB6

CNB7

CNB8

CNB9
CNB10