The Human Nucleolar Protein FTSJ3 Associates with NIP7 and Functions in Pre-rRNA Processing

NIP7 is one of the many trans-acting factors required for eukaryotic ribosome biogenesis, which interacts with nascent pre-ribosomal particles and dissociates as they complete maturation and are exported to the cytoplasm. By using conditional knockdown, we have shown previously that yeast Nip7p is required primarily for 60S subunit synthesis while human NIP7 is involved in the biogenesis of 40S subunit. This raised the possibility that human NIP7 interacts with a different set of proteins as compared to the yeast protein. By using the yeast two-hybrid system we identified FTSJ3, a putative ortholog of yeast Spb1p, as a human NIP7-interacting protein. A functional association between NIP7 and FTSJ3 is further supported by colocalization and coimmunoprecipitation analyses. Conditional knockdown revealed that depletion of FTSJ3 affects cell proliferation and causes pre-rRNA processing defects. The major pre-rRNA processing defect involves accumulation of the 34S pre-rRNA encompassing from site A′ to site 2b. Accumulation of this pre-rRNA indicates that processing of sites A0, 1 and 2 are slower in cells depleted of FTSJ3 and implicates FTSJ3 in the pathway leading to 18S rRNA maturation as observed previously for NIP7. The results presented in this work indicate a close functional interaction between NIP7 and FTSJ3 during pre-rRNA processing and show that FTSJ3 participates in ribosome synthesis in human cells

Structural Characterization of the Internal Transcribed Spacer 2 (ITS2) of the Ribosomal DNA (rDNA) Cluster in Calyptratae (Diptera: Schizophora) and its Implications for Molecular Phylogenetic Analyses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)The internal transcribed spacer 2 (ITS2) of the eukaryotic ribosomal DNA (rDNA) cluster plays an essential role in processing of the ribosomal RNA, which is primarily accomplished by the secondary structures acquired by the molecule after transcription. Two possible structural conformation models have been proposed for the ITS2 region, the 'ring model' and the 'hairpin model,' and the former has been widely used in many molecular phylogenetic analyses incorporating structural information available to date. To evaluate the validity of this model, in vitro transcribed ITS2 molecules from species representing the three superfamilies of the Calyptratae clade (Diptera: Schizophora), namely Cochliomyia hominivorax, Musca domestica, and Glossina morsitans, were submitted to enzymatic digestion with single- and double-stranded specific nucleases (RNases I, A, T1, and V1). The resulting fragments were analyzed by capillary electrophoresis and digestion sites were mapped in the secondary structure models which were obtained by in silico prediction with further refinement by homology comparisons. The pattern of RNA fragments generated by these RNases show a high degree of correlation to most of the predicted helix-loop regions and structural motifs. Discrepancies to the models can be explained by alternative structural conformation dynamics (in M. domestica and G. morsitans) and by higher-order factors (such as tertiary interactions) that may stabilize thermodynamically unfavored structures (in C. hominivorax).763158171Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [06/61217-3

Identification and Characterization of an Alternatively Spliced Isoform of the Human Protein Phosphatase 2A alpha Catalytic Subunit

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)PP2A is the main serine/threonine-specific phosphatase in animal cells. The active phosphatase has been described as a holoenzyme consisting of a catalytic, a scaffolding, and a variable regulatory subunit, all encoded by multiple genes, allowing for the assembly of more than 70 different holoenzymes. The catalytic subunit can also interact with alpha 4, TIPRL (TIP41, TOR signaling pathway regulator-like), the methyl-transferase LCMT-1, and the methyl-esterase PME-1. Here, we report that the gene encoding the catalytic subunit PP2Ac alpha can generate two mRNA types, the standard mRNA and a shorter isoform, lacking exon 5, which we termed PP2Ac alpha 2. Higher levels of the PP2Ac alpha 2 mRNA, equivalent to the level of the longer PP2Ac alpha mRNA, were detected in peripheral blood mononuclear cells that were left to rest for 24 h. After this time, the peripheral blood mononuclear cells are still viable and the PP2Ac alpha 2 mRNA decreases soon after they are transferred to culture medium, showing that generation of the shorter isoform depends on the incubation conditions. FLAG-tagged PP2Ac alpha 2 expressed in HEK293 is catalytically inactive. It displays a specific interaction profile with enhanced binding to the alpha 4 regulatory subunit, but no binding to the scaffolding subunit and PME-1. Consistently, alpha 4 out-competes PME-1 and LCMT-1 for binding to both PP2Ac alpha isoforms in pulldown assays. Together with molecular modeling studies, this suggests that all three regulators share a common binding surface on the catalytic sub-unit. Our findings add important new insights into the complex mechanisms of PP2A regulation.287748534862Fundacao de Amparo a Pesquisa do Estado Sao PauloConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Laboratorio Nacional de Biociencias, Centro Nacional de Pesquisa em Energia e MateriaisConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Evidence for the association of the human regulatory protein Ki-1/57 with the translational machinery

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Ki-1/57 is a cytoplasmic and nuclear protein of 57 kDa first identified in malignant cells from Hodgkin's lymphoma. Based on yeast-two hybrid protein interaction we found out that Ki-1/57 interacts with adaptor protein RACK1 (receptor of activated kinase 1), CIRP (cold-inducible RNA-binding protein), RPL38 (ribosomal protein L38) and FXR1 (fragile X mental retardation-related protein 1). Since these proteins are involved in the regulation of translation we suspected that Ki-1/57 may have a role in it. We show by immunoprecipitation the association of Ki-1/57 with FMRP. Confocal microscopy revealed that Ki-1/57 colocalizes with FMRP/FXR1/2 to stress granules. Furthermore Ki-1/57 cosediments with free ribosomal particles and enhances translation, when tethered to a reporter mRNA, suggesting that Ki-1/57 may be involved in translational regulation. Structured summary of protein interactions: Ki-1/57 and TIA-1 colocalize by fluorescence microscopy (View interaction) Ki-1/57 physically interacts with CIRP by two hybrid (View interaction) FMRP physically interacts with Ki-1/57 by anti bait coimmunoprecipitation (View interaction) Ki-1/57 physically interacts with FXR1 by two hybrid (View interaction) Ki-1/57 physically interacts with RPL38 by two hybrid (View interaction) (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.5851625562560Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)LNBio/CNPEMFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Increased CCL2 and IL-8 in the Bone Marrow Microenvironment in Acute Lymphoblastic Leukemia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Background. The interactions of acute lymphoblastic leukemia (ALL) blasts with bone marrow (BM) stromal cells have a positive impact on leukemia cell survival. In the present study, we proposed to identify and investigate the role of molecules critically involved in leukemia-microenvironment crosstalk. Procedure. Gene expression profiling analyses of BM mesenchymal stem cells (BMMSC) were performed following stimulation by ALL cells. CCL2 and IL-8 plasma levels were evaluated from, ALL patients and controls. Expression of the CCL2 and IL-8 receptors in ALL was determined by RT-PCR. The biological effects of CCL2, IL-8 or its neutralizing antibodies in primary precursor-B ALL and BMMSC cells were evaluated using in vitro assays. Results. Leukemia stimulation of BMMSC upregulated the expression of several inflammatory chemokines, including CCL2 and IL-8. The BM plasma levels of CCL2 and IL-8 in children at diagnosis were significantly higher than in healthy controls (P < 0.00)). Functional studies revealed that CCL2 and IL-8 enhanced the capacity of BMMSC to support adhesion of ALL cells. CCL2 and IL-8 were also found to enhance BMMSC survival and to increase their proliferation. ALL cells were not directly affected by CCL2 or IL-8. Conclusions. The leukemic BM microenvironment had increased levels of CCL2 and IL-8. These chemokines are known to have suppressive effects in normal hematopoiesis. Our data indicate that CCL2 and IL-8 have a positive impact on BMMSC survival, proliferation, and adhesiveness to ALL cells. Leukemia-associated CCL2 and IL-8 upregulation may represent one possible mechanism of microenvironment perversion in favor of ALL cells. Pediatr Blood Cancer 2011;56:568-577. (C) 2010 Wiley-Liss, Inc.564568577Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [05/02390-4, 06/02083-7, 06/01158-3, 08/02106-2]CNPq [401122/05, 382.687/2008-6