23 research outputs found
Role of Rrp23 in the Formation of Ribosomal Subunits
[ES]Durante la formación de las subunidades ribosómicas pequeñas, los complejos pre-40S que se forman en el nucleolo son transportados rápidamente al citoplasma, el lugar donde completan su maduración. Los eventos que suceden durante el tránsito de dichos complejos por el nucleoplasma y los mecanismos que median su exportación son todavía muy pocos conocidos. En esta tesis nos propusimos aportar nueva información sobre estos procesos mediante el estudio de Rrp12, un factor que se asocia a los complejos pre-40S dentro del núcleo. El estudio se realizó en la levadura Saccharomyces cerevisiae, el organismo más utilizado para estudiar la ruta de síntesis de ribosomas.
Hemos demostrado que Rrp12 es un factor necesario para el transporte de complejos pre-40S del núcleo al citoplasma, y para una maduración eficiente de pre-ribosomas 90S, los complejos que preceden a los pre-40S en la ruta. Así, la eliminación de Rrp12 causa una acumulación de pre-ribosomas 90S tardíos, un procesamiento anormal del pre-rRNA 35S, un retraso en la eliminación de fragmentos subproducto del procesamiento, y un bloqueo en la exportación de partículas pre-40S nucleares. También hemos demostrado que la exportina Crm1/Xpo1 es necesaria para los mismos pasos de maduración en los que interviene Rrp12. Por lo tanto, además de participar en la exportación, Rrp12 y Crm1 son necesarias para pasos de la ruta que suceden en el nucleolo y que son anteriores al paso de exportación. Los resultados obtenidos indican que, en la ruta de síntesis de subunidades 40S, el ensamblaje de partículas pre-40S, la degradación de productos de procesamiento, y la adquisición de competencia para la exportación nuclear tienen lugar de forma integrada, en un paso común, en partículas pre-ribosómicas 90S tardías.[EN]During the synthesis of small ribosomal subunits in eukaryotes, the pre‐40S
particles formed in the nucleolus are rapidly transported to the cytoplasm. The
mechanisms involved in the nuclear export of these particles and its coordination
with other steps of the 40S synthesis pathway are mostly unknown. This thesis
focused on the study of Rrp12, an evolutionary‐conserved protein previously found
to be present in nuclear pre‐40S particles. The initial finding of the study was that
the conditional depletion of Rrp12 in yeast impairs the production of 40S, but not
60S, ribosomal subunits. A detailed analysis of the depletion phenotype, using a
combination of genetic, biochemical, cell‐biology and proteomic approaches,
unveiled that Rrp12 is specifically required for the exit of pre‐40S particles to the
cytoplasm. In addition, it was found that Rrp12, together with the Crm1/Xpo1
exportin, participates in processes that occur in early pre‐ribosomes in the
nucleolus, including the processing of the pre‐rRNA and the elimination of
processing byproducts. Thus, the two pre‐40S export factors Rrp12 and Crm1/Xpo1
participate in maturation steps that take place in the nucleolus, upstream of nuclear
export. Altogether, the findings of this work indicate that, in the 40S subunit
synthesis pathway, the completion of early pre‐40S particle assembly, the initiation
of byproduct degradation and the priming for nuclear export occur in an integrated
manner in nucleolar pre‐ribosomes
Rrp12 and the Exportin Crm1 Participate in Late Assembly Events in the Nucleolus during 40S Ribosomal Subunit Biogenesis
This is an open-access article distributed under the terms of the Creative Commons Attribution License.During the biogenesis of small ribosomal subunits in eukaryotes, the pre-40S particles formed in the nucleolus are rapidly transported to the cytoplasm. The mechanisms underlying the nuclear export of these particles and its coordination with other biogenesis steps are mostly unknown. Here we show that yeast Rrp12 is required for the exit of pre-40S particles to the cytoplasm and for proper maturation dynamics of upstream 90S pre-ribosomes. Due to this, in vivo elimination of Rrp12 leads to an accumulation of nucleoplasmic 90S to pre-40S transitional particles, abnormal 35S pre-rRNA processing, delayed elimination of processing byproducts, and no export of intermediate pre-40S complexes. The exportin Crm1 is also required for the same pre-ribosome maturation events that involve Rrp12. Thus, in addition to their implication in nuclear export, Rrp12 and Crm1 participate in earlier biosynthetic steps that take place in the nucleolus. Our results indicate that, in the 40S subunit synthesis pathway, the completion of early pre-40S particle assembly, the initiation of byproduct degradation and the priming for nuclear export occur in an integrated manner in late 90S pre-ribosomes.This work is supported by grants from both the Spanish Ministry of Economy and Competitiveness (BFU2011-23668, RD06/0020/0001 and RD12/0036/0002), the Samuel Solórzano Barruso Foundation (FS/17-2013) and the Castilla y León Autonomous Government (CSI039A12-1). GM and BN have been supported by graduate student contracts by the University of Salamanca and Santander Bank and, in the case of BN, by the RD06/0020/0001 grant. Spanish funding is co-sponsored by the European Union FEDER Program.Peer Reviewe
Contribution of the R-Ras2 GTP-binding protein to primary breast tumorigenesis and late-stage metastatic disease
R-Ras2 is a transforming GTPase that shares downstream effectors with Ras subfamily proteins. However, little information exists about the function of this protein in tumorigenesis and its signalling overlap with classical Ras GTPases. Here we show, by combining loss- and gain-of-function studies in breast cancer cells, mammary epithelial cells and mouse models, that endogenous R-Ras2 has a role in both primary breast tumorigenesis and the late metastatic steps of cancer cells in the lung parenchyma. R-Ras2 drives tumorigenesis in a phosphatidylinostiol-3 kinase (PI3K)-dependent and signalling autonomous manner. By contrast, its prometastatic role requires other priming oncogenic signals and the engagement of several downstream elements. R-Ras2 function is required even in cancer cells exhibiting constitutive activation of classical Ras proteins, indicating that these GTPases are not functionally redundant. Our results also suggest that application of long-term R-Ras2 therapies will result in the development of compensatory mechanisms in breast tumoursFil: Larive, Ramon. Universidad de Salamanca; España. University of Montpellier I and II; FranciaFil: Moriggi, Giulia. Universidad de Salamanca; EspañaFil: Menacho Márquez, Mauricio Ariel. Universidad de Salamanca; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Cañamero, Marta. Centro Nacional de Investigaciones Oncológicas; EspañaFil: de Alava, Enrique. Universidad de Salamanca; España. Hospital Universitario Virgen del Rocío. Sevilla; EspañaFil: Alarcón, Balbino. Centro de Biología Molecular ‘‘Severo Ochoa’. Madrid; EspañaFil: Dosil, Mercedes. Universidad de Salamanca; EspañaFil: Bustelo, Xosé R.. Universidad de Salamanca; Españ
Statin or fibrate chronic treatment modifies the proteomic profile of rat skeletal muscle
Identification of an extranucleolar site of ribosome production in yeast
Póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.In budding yeast the nucleolus is a crescent-shaped structure that abuts the nuclear envelope and occupies up to one-third of the nucleus. In mitosis, the nucleolus remains intact and splits just when the rDNA is segregated, at the end of anaphase. Thus, the nucleolar ribosome synthesis machinery
persists as a recognizable region throughout mitosis and streams from the mother into the daughter during telophase. We have unveiled that in early mitosis there is a site of ribosome production that is separate from the bulk of the nucleolar material. We found that, during a short time in metaphase,
several ribosome-maturation proteins are present both in the nucleolus and at a punctate body located in the vicinity of the nuclear envelope in the emerging daughter cell. Such body is well-separated from the bulk of the rDNA, which is mostly located in the mother cell, and also from the daughter spindle pole body. It is a body that contains rDNA-binding proteins, pre-rRNAs and ribosome maturation factors characteristic of nucleolar and nucleoplasmic pre-ribosomes, but is devoid of late maturation factors. The focal accumulation of pre-export 40S subunits at this discrete site is being used as a tool to study when and how different factors are incorporated into the 40S subunit synthesis pathway.This work is supported by grants from the spanish Ministerio de Economía y Competitividad (BFU2011-23668, BFU2014-52729, and RD12/0036/0002), the Samuel Solórzano Barruso Foundation (FS/17-2013) and the Castilla y León Autonomous Government (CSI039A12-1). G.M. and B.N. have been supported by graduate student contracts by the University of Salamanca and Santander Bank and, in the case of B.N., by the RD06/0020/0001 grant. Spanish funding is co-sponsored by the European Union FEDER Program.Peer reviewe
Rrp12 is not involved in pre-40S particle assembly.
<p>(<b>A</b> to <b>C</b>) Bottom panels, Northern blot analysis showing coimmunoprecipitation of the 20S pre-rRNA with Enp1-GFP (A), Dim1-MYC (B), Tsr1-GFP (C), Ltv1-GFP (C), Rio2-GFP (C), Nob1-GFP (C) and Nop7-GFP (C) before (0) and upon depletion of Rrp12 for 9 hours. Top panels, Western blot analysis showing the amount of immunoprecipitated proteins in these experiments. Mobility of pre-RNA species is indicated on the left of each bottom panel. Antibodies used in the immunoblots and Northern blot probes are shown on the right of the top and bottom panels, respectively. The thin white lines between lanes 3 and 4, and 9 and 10, shown in A and B, indicate the presence of in-between lanes in the same blot that have been removed. (<b>D</b>) Western blot analyses of trichloroacetic acid (TCA) precipitated cell lysates showing the amount of Rrp12 (top panels) and the indicated GFP-tagged proteins (middle panels) under the indicated growth conditions. The amount of Cdc11 was used as loading control (bottom panel). (<b>E</b>) Pre-ribosomal factors (listed on the left) copurifying with the indicated GFP-tagged proteins (top) in the presence (columns 1 to 4) or absence (columns 5 to 8) of Rrp12. Copurification of a factor with the bait is indicated with a dot. For Rrp12 depletion, <i>GAL::HA-rrp12</i> cells were shifted from galactose-containing media to glucose-containing media for 12 hours. The pre-ribosomal particles that contain the prey proteins are indicated on the left. Size of dots represents the relative amount of coimmunoprecipitated protein in each case (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004836#s4" target="_blank">Materials and Methods</a>).</p
Rrp12 and the Exportin Crm1 Participate in Late Assembly Events in the Nucleolus during 40S Ribosomal Subunit Biogenesis
<div><p>During the biogenesis of small ribosomal subunits in eukaryotes, the pre-40S particles formed in the nucleolus are rapidly transported to the cytoplasm. The mechanisms underlying the nuclear export of these particles and its coordination with other biogenesis steps are mostly unknown. Here we show that yeast Rrp12 is required for the exit of pre-40S particles to the cytoplasm and for proper maturation dynamics of upstream 90S pre-ribosomes. Due to this, in vivo elimination of Rrp12 leads to an accumulation of nucleoplasmic 90S to pre-40S transitional particles, abnormal 35S pre-rRNA processing, delayed elimination of processing byproducts, and no export of intermediate pre-40S complexes. The exportin Crm1 is also required for the same pre-ribosome maturation events that involve Rrp12. Thus, in addition to their implication in nuclear export, Rrp12 and Crm1 participate in earlier biosynthetic steps that take place in the nucleolus. Our results indicate that, in the 40S subunit synthesis pathway, the completion of early pre-40S particle assembly, the initiation of byproduct degradation and the priming for nuclear export occur in an integrated manner in late 90S pre-ribosomes.</p></div
Rrp12 is present in both 90S pre-ribosomes and pre-40S particles.
<p>(<b>A</b>) Scheme of the maturation of pre-ribosomes. The names of specific factors frequently used for purifying each pre-ribosome are indicated on the right. In rapidly growing cells, ∼60% of primary transcripts are cleaved at A<sub>0</sub>–A<sub>1</sub>–A<sub>2</sub> co-transcriptionally within the small subunit (SSU) processome and, after this, the precursor of the large subunit (pre-LSU) is assembled onto the nascent pre-rRNA. When not cleaved co-transcriptionally, the full-length 35S pre-rRNA is assembled into the 90S pre-ribosome, a particle very similar to the SSU-processome. The order of incorporation of the seven major maturation factors present in cytoplasmic pre-40S particles is shown on the left. Enp1, Dim1 and Pno1 are recruited to 90S/SSU particles. Tsr1 is recruited to early pre-40S particles in the nucleolus. Ltv1 and Nob1 join pre-40S particles in the nucleus. The step of incorporation of Rio2 remains ill defined. (<b>B</b>) Western blot analysis showing coimmunoprecipitation of Rrp12 (second panels from top) and of the control protein Rpl1 (bottom panels) with the indicated 90S pre-ribosome and nuclear pre-40S factors (top) in the presence (+) or absence (−) of RNase A in cell lysates. Factors present in 90S, pre-40S and pre-60S particles are shaded in brown, blue and green, respectively. The amount of GFP-Trap purified bait is shown in the first panels from top. The asterisk indicates a protein species in the Enp1-GFP purification lane that probably corresponds to a partial degradation product. (<b>C</b> and <b>D</b>) Northern blot analysis showing coimmunoprecipitation of pre-rRNA species (second to bottom panels on the right) with the indicated MYC-tagged proteins in normal cells. As control, parallel Northern blots were performed on total RNAs prepared from the same total cell lysate samples used for the immunoprecipitations (second to bottom panels on the left). Western blot experiments were performed to analyze the amount of MYC-tagged protein present in the total cell lysates (top panel on the left) and immunoprecipitations (top panel on the right). TCL, total cell lysates. IP, immunoprecipitation.</p
Model for the integration of different processes in the nucleolus during synthesis of 40S subunits.
<p>The 90S pre-ribosome contains ∼70 factors (represented in orange) that are specifically required for the cleavage of the primary pre-rRNA at sites A<sub>0</sub>, A<sub>1</sub> and A<sub>2</sub>, and for the assembly of ribosomal proteins (not represented). In addition, the 90S pre-ribosome engages two other sets of proteins that participate in activities that will be initiated at the time of, or immediately after, the A<sub>2</sub> cleavage: the exosome complex, and Rrp12/Crm1. The exosome degrades the 5′-A<sub>0</sub> fragment, allowing the liberation and recycling of bound 90S proteins. Rrp12 and Crm1 act as export factors for the released pre-40S particle. The cleavage of the pre-rRNA at site A<sub>2</sub> is intertwined with the initiation of 5′-A<sub>0</sub> degradation and the priming of the emergent pre-40S particle for nuclear export. During the rapid transit of the pre-40S particle from the nucleolus to the cytoplasm, a few maturation factors (Tsr1, Rio2, Nob1) that will be required in the cytoplasm are incorporated in a manner independent of nuclear export. Another maturation factor, Ltv1, requires Rrp12 for its stable incorporation onto pre-40S particles, but whether or not it is dependent on the export process itself remains to be ascertained. Further details about this model, and the evidence supporting it, is given in the text.</p
Loss of Rrp12 causes accumulation of 5′-A<sub>0</sub>-containing 90S pre-ribosomes.
<p>(<b>A</b>) Top panel, sucrose-gradient sedimentation analysis of ribosomal fractions (40S, 60S, 80S and polysomes) of cell lysates from the control wild type strain grown in glucose-containing media, and the <i>GAL::HA-rrp12</i> strain grown in galactose-containing media and shifted to glucose-containing media for 9 hours. Bottom panels, Northern (second to sixth panels from top) and Western (bottom panel) blot analyses of indicated components of pre-ribosomal particles in fractions obtained in the gradients. Numbers of fractions are shown at the bottom. Blotting probes and antibodies are indicated on the right. (<b>B</b>) Northern blot analysis showing copurification (second to fourth panels on the right) of the indicated pre-RNA species, U3 snoRNA and 5′-A<sub>0</sub> fragment with Pwp2-GFP in the indicated yeast strains and culture conditions (top). As control, parallel Northern blots were performed on total RNAs prepared from the same total cell lysate samples used for the immunoprecipitations (second to third panels on the left). Western blot experiments were performed to analyze the amount of Pwp2-GFP present in the total cell lysates (top panel on the left) and GFP-Trap purified complexes (top panel on the right). Asterisks indicate pre-rRNA species that do not correspond to any major processing intermediate, which probably are 35S partial degradation products. (<b>C</b>) Sucrose gradient analysis showing the sedimentation behavior of Pwp2-GFP and Rps8 in the presence (top two panels) and absence (bottom two panels) of Rrp12. The positions of the gradient where 40S, 60S and 80S complexes sedimented are indicated by arrows. (<b>D</b>) Top panels, epifluorescence microscopy analysis of the subcellular distribution of Pwp2-GFP before (top left panel) and upon depletion (top right panel) of Rrp12. Bottom panels, DIC images of above preparations.</p