Biomedical subjects

Beatriz M A Fontoura

Publications and source records attributed to Beatriz M A Fontoura.

6 recordsLinked to original sources

PubMed2006

The nucleoporin Nup96 is required for proper expression of interferon-regulated proteins and functions.

Nup98 and Nup96 are components of the nuclear transport machinery and are induced by interferons (IFN). Nup98 is a constituent of an mRNA export pathway that is targeted by viruses and regulated by IFN. However, the role of Nup96 in IFN-related mechanisms has not been established. To investigate the function of Nup96 in vivo, we generated Nup96(+/-) mice that express low levels of Nup96, as Nup96(-/-) mice are lethal. The Nup96(+/-) mice presented selective alterations of the immune system, which resulted in downregulation and impaired IFN alpha- and gamma-mediated induction of MHC I and IFNgamma induction of MHC II, ICAM-1, and other proteins. Frequency of TCRalphabeta+ and CD4+ T cells, which depends on MHC function, is reduced in NUP96(+/-) mice. Upon immunization, Nup96(+/-) mice showed impaired antigen presentation and T cell proliferation. Nup96(+/-) cells and mice were highly susceptible to viral infection, demonstrating a role for Nup96 in innate and adaptive immunity.

Animals↗

PubMed2006

Nuclear export assays for poly(A) RNAs.

Nuclear export of mRNAs is a central step in eukaryotic gene expression. A defect in bulk poly(A) RNA export can be caused either by a direct disruption of the mRNA export machinery or by an indirect effect on mRNA biogenesis. One example of interference with the mRNA export pathway is viral-host interactions involving mRNA export factors. VSV M protein binds the mRNA export factor Rae1 that is in complex with Nup98, resulting in nuclear retention of mRNAs. To study regulation of mRNA export, we review here two useful methodologies, which include a reporter gene assay and oligo(dT) in situ hybridization. In a reporter gene assay one can assess up-regulation or down-regulation of gene expression that can occur at different levels, including transcription, mRNA processing, mRNA export, and translation. An effect on mRNA export can then be identified by determining the intracellular distribution of poly(A) RNA using oligo(dT) in situ hybridization. Reporter gene assays are quick, relatively simple and can thus be used in primary highthroughput screenings. To further pinpoint disruption of mRNA export, oligo(dT) in situ hybridization can be used. Since it is a more laborious methodology it is more suitable for a secondary screening. We also review here a combination of oligo(dT) in situ hybridization with immunofluorescence for simultaneous localization of endogenous or ectopically expressed proteins. Altogether, these assays are valuable tools for identifying major regulatory effects on mRNA nuclear export.

Active Transport, Cell Nucleus↗

PubMed2005

VSV disrupts the Rae1/mrnp41 mRNA nuclear export pathway.

Interference with nucleocytoplasmic transport is a strategy employed by certain viruses to compromise host cellular function. While it has been shown that the matrix (M) protein of the vesicular stomatitis virus (VSV) inhibits nuclear export of host cell mRNAs, the underlying mechanism has not been fully established. Here we show that VSV M protein binds the mRNA export factor Rae1/mrnp41. A mutant of M protein defective in Rae1 binding is unable to inhibit mRNA nuclear export. We further show that increased expression of Rae1 fully reverts the inhibition of mRNA export induced by M protein or following virus infection. We found that Rae1 is induced by interferon-gamma, a cytokine that plays a critical role in the immune response to viruses, such as VSV. Thus, these results demonstrate that VSV M protein blocks mRNA export by disrupting Rae1 function, which can be reverted by induction of Rae1 expression.

Active Transport, Cell Nucleus↗

PubMed2003

Cytoplasmic complex of p53 and eEF2.

We have shown previously that cytoplasmic p53 is covalently linked to 5.8S rRNA. The covalent complex is associated with a small subset of polyribosomes, which includes polyribosomes translating p53 mRNA. Because 5.8S rRNA resides in or near the ribosomal P site, our findings suggested involvement of p53 in translational regulation. Ninety-seven kiloDaltons eEF2 was found to coimmunoprecipitate in a salt-stable complex with p53. The 97 kDa species was identified as eEF2, because it was (1) recognized by a polyclonal antiserum specific for eEF2, (2) ADP-ribosylated by diphtheria toxin (DT), and (3) radiolabeled by gamma-32P-azido-GTP and UV-irradiation. p53 and eEF2 sedimented in sucrose gradients in both polyribosomal and subribosomal fractions. Subribosomal p53 can bind eEF2 without the mediation of ribosomes, because (1) it binds subribososomal eEF2, (2) it binds phosphorylated eEF2, and (3) subribosomal p53-bound eEF2 can be ADP-ribosylated by DT. No effect of p53 activation was found on eEF2 expression or phosphorylation. However, the binding of eEF2 to p53 decreased when cytoplasmic p53 migrated to the nucleus. Renaturation of temperature sensitive A135V mutant p53 (ts-p53) was found to alter the sensitivity of p53 mRNA translation, but not bulk mRNA translation, to the translocation-specific elongation inhibitor, cycloheximide (Cx). The association of p53 with two translational components involved in ribosomal translocation, eEF2 and 5.8S rRNA, and the effect of p53 on sensitivity to the translocation inhibitor, Cx, as well as the known molecular interactions of these components in the ribosome suggest involvement of p53 in elongation.

Animals↗

PubMed2003

Sec13 shuttles between the nucleus and the cytoplasm and stably interacts with Nup96 at the nuclear pore complex.

Sec13 is a constituent of the endoplasmic reticulum and the nuclear pore complex (NPC). At the endoplasmic reticulum, Sec13 is involved in the biogenesis of COPII-coated vesicles, whereas at the NPC its function is unknown. We show here, by yeast two-hybrid screenings and biochemical assays, that a region at the amino terminus of the human nuclear pore complex protein Nup96 interacts with the WD (Trp-Asp) repeat region of human Sec13. By using immunofluorescence and confocal and immunoelectron microscopy, we found that in interphase, Sec13 and Nup96 are localized at both sides of the NPC in addition to other intracellular sites. In mitosis, Sec13 was found dispersed throughout the cell, whereas a pool of Nup96 colocalized with the spindle apparatus. Photobleaching experiments showed that Sec13 shuttles between intranuclear sites and the cytoplasm, and a fraction of Sec13 is stably associated with NPCs. Cotransfection of Sec13 and the Sec13 binding site of Nup96 decreased the mobile pool of Sec13, demonstrating the interaction of Sec13 and Nup96 in vivo. Targeting studies showed that Sec13 is actively transported into the nucleus and contains a nuclear localization signal. These results indicate that Sec13 stably interacts with Nup96 at the NPC during interphase and that the shuttling of Sec13 between the nucleus and the cytoplasm may couple and regulate functions between these two compartments.

Active Transport, Cell Nucleus↗

PubMed2002

Role of nucleoporin induction in releasing an mRNA nuclear export block.

Signal-mediated nuclear import and export proceed through the nuclear pore complex (NPC). Some NPC components, such as the nucleoporins (Nups) Nup98 and Nup96, are also associated with the nuclear interior. Nup98 is a target of the vesicular stomatitis virus (VSV) matrix (M) protein-mediated inhibition of messenger RNA (mRNA) nuclear export. Here, Nup98 and Nup96 were found to be up-regulated by interferon (IFN). M protein-mediated inhibition of mRNA nuclear export was reversed when cells were treated with IFN-gamma or transfected with a complementary DNA (cDNA) encoding Nup98 and Nup96. Thus, increased Nup98 and Nup96 expression constitutes an IFN-mediated mechanism that reverses M protein-mediated inhibition of gene expression.

Active Transport, Cell Nucleus↗