CPEBs studies on the cell cycle : mapping CPEBs network and unveiling a new function for CPEB-mediated cap-ribose methylation

En el capitulo I mostramos la caracterización funcional de la ribosa-metiltransferasa de cap I en Xenopus laevis. Se trata de una enzima localizada tanto en el núcleo como en el citoplasma y que tras la estimulación con progesterona de los ovocitos interacciona con CPEB1 de una manera dependiente del ARNm. Asimismo, mostramos que la modificación del cap realizada por ella es necesaria para la activación de la traducción de un ARNm reportero en mayor medida que la elongación de la cola de poly(A). En el capitulo II hemos llevado a cabo la caracterización funcional de CPEB1, CPEB2 y CPEB4 durante el ciclo celular. Hemos encontrado que CPEB1 es necesaria para el progreso de la fase S, la proliferación celular, los anclajes célulamatriz y las primeras fases de mitosis (profase). CPEB2 actúa después de CPEB1, siendo necesaria durante metafase, mientras que CPEB4 es requerida durante la última etapa de mitosis y citoquinesis. Asímismo, hemos descubierto que CPEB1, CPEB2 y CPEB4 están interconectadas durante la progresión del ciclo celular, de tal modo que los niveles y actividades relativas de cada una están estrictamente reguladas. En conclusión, estos resultados avanzan otro paso en la compresión de la función de las CPEBs en la regulación del ciclo celular, desvelando una red regulatoria de las CPEBs durante la progesión del ciclo celular en células somáticas.In the chapter I, we characterized the functional cap I ribose methyltransferase in Xenopus laevis. This enzyme is a nucleo-cytoplasmic shuttling protein, which interacts with CPEB1 upon progesterone stimulation in a RNA-dependent manner. The modification of the cap is required for the translational activation of a reporter mRNA, more than the elongation of the poly(A)tail. In the chapter II, we narrowed down the functions of CPEB1, CPEB2 and CPEB4 during the cell cycle. We found that CPEB1 is needed for proper S phase, cell proliferation, cell-to-matrix attachment and for early steps of mitosis (prophase). In mitosis CPEB2 functions after CPEB1, being needed in metaphase, while CPEB4 is required for the last step of mitosis and cytokinesis. Moreover we found that CPEB1, CPEB2 and CPEB4 are interconnected during somatic cell cycle progression, showing that their relative levels and activities are tightly regulated to accomplish proper cell division. Altogether these results add another step in the understanding of CPEBs role during the cell cycle, unveiling a new map in the CPEBs network during somatic cell cycle progression

CPEBs studies on the cell cycle : mapping CPEBs network and unveiling a new function for CPEB-mediated cap-ribose methylation

En el capitulo I mostramos la caracterización funcional de la ribosa-metiltransferasa de cap I en Xenopus laevis. Se trata de una enzima localizada tanto en el núcleo como en el citoplasma y que tras la estimulación con progesterona de los ovocitos interacciona con CPEB1 de una manera dependiente del ARNm. Asimismo, mostramos que la modificación del cap realizada por ella es necesaria para la activación de la traducción de un ARNm reportero en mayor medida que la elongación de la cola de poly(A). En el capitulo II hemos llevado a cabo la caracterización funcional de CPEB1, CPEB2 y CPEB4 durante el ciclo celular. Hemos encontrado que CPEB1 es necesaria para el progreso de la fase S, la proliferación celular, los anclajes célulamatriz y las primeras fases de mitosis (profase). CPEB2 actúa después de CPEB1, siendo necesaria durante metafase, mientras que CPEB4 es requerida durante la última etapa de mitosis y citoquinesis. Asímismo, hemos descubierto que CPEB1, CPEB2 y CPEB4 están interconectadas durante la progresión del ciclo celular, de tal modo que los niveles y actividades relativas de cada una están estrictamente reguladas. En conclusión, estos resultados avanzan otro paso en la compresión de la función de las CPEBs en la regulación del ciclo celular, desvelando una red regulatoria de las CPEBs durante la progesión del ciclo celular en células somáticas.In the chapter I, we characterized the functional cap I ribose methyltransferase in Xenopus laevis. This enzyme is a nucleo-cytoplasmic shuttling protein, which interacts with CPEB1 upon progesterone stimulation in a RNA-dependent manner. The modification of the cap is required for the translational activation of a reporter mRNA, more than the elongation of the poly(A)tail. In the chapter II, we narrowed down the functions of CPEB1, CPEB2 and CPEB4 during the cell cycle. We found that CPEB1 is needed for proper S phase, cell proliferation, cell-to-matrix attachment and for early steps of mitosis (prophase). In mitosis CPEB2 functions after CPEB1, being needed in metaphase, while CPEB4 is required for the last step of mitosis and cytokinesis. Moreover we found that CPEB1, CPEB2 and CPEB4 are interconnected during somatic cell cycle progression, showing that their relative levels and activities are tightly regulated to accomplish proper cell division. Altogether these results add another step in the understanding of CPEBs role during the cell cycle, unveiling a new map in the CPEBs network during somatic cell cycle progression

Global Analysis of CPEBs Reveals Sequential and Non-Redundant Functions in Mitotic Cell Cycle

<div><p>CPEB (Cytoplasmic Polyadenylation Element Binding) proteins are a family of four RNA-binding proteins that regulate the translation of maternal mRNAs controlling meiotic cell cycle progression. But CPEBs are not limited to the transcriptionally silent germline; they are also expressed, in various combinations, in somatic cells, yet their role in regulation of mitosis-related gene expression is largely unknown. Deregulation of CPEB1 and CPEB4 have been linked to tumor development. However, a systematic analysis addressing their requirements for the temporal regulation of mitotic gene expression has yet to be performed. This study addresses the requirements of each of the four CPEBs for mitotic phase transitions, with a particular focus on cytoplasmic polyadenylation and translational regulation. We demonstrate that CPEB3 is the only member dispensable for mitotic cell division, whereas the other three members, CPEB1, 2, and 4, are essential to successful mitotic cell division. Thus, CPEB1 is required for prophase entry, CPEB2 for metaphase and CPEB4 for cytokinesis. These three CPEBs have sequential non-redundant functions that promote the phase-specific polyadenylation and translational activation of CPE-regulated transcripts in the mitotic cell cycle.</p></div

CPEB1 is required for prophase entry; CPEB2 for metaphase-to-anaphase transition and CPEB4 for cytokinesis.

<p>(A) HEK-293 cells stably expressing an IPTG-inducible system for each CPEB knock-down were induced or not with IPTG. Two days after induction, cells were transfected with a plasmid encoding fluorescent histon H2B. After one additional day cells were recorded by live imaging experiments. Images were acquired every 10 minutes and analyzed for mitotic progression. Representative images are shown for individual cells in interphase or during specific phases of mitosis that are readily identified by chromosome condensation state and organisation. (B) Mitotic-stage analysis of 50 cells from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138794#pone.0138794.s002" target="_blank">S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138794#pone.0138794.s006" target="_blank">S5</a> Videos as in Fig 2A. On Y axis, each lane represents one cell. On X axis, time is represented as minutes. Colors represent the indicated mitotic phases. Mitotic entry was determined by analyzing the first signs of DNA condensation cross-reinforced with cell-rounding. Mitotic exit was scored based on chromosome segregation at anaphase and DNA decondensation. Sh, short-hairpin; CTRL, control.</p

CPEB1, CPEB2 and CPEB4 are required for proper GFP translation at G2 and M phases.

<p>(A) HEK-293 cells stably expressing an IPTG-inducible system for each CPEB knock-down and carrying a GFP-3'UTR +/-CPE together with a RFP-3'UTR were induced or not with IPTG. Two days after induction, cells were recorded by live imaging experiments and analyzed for GFP expression (see Fig I in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138794#pone.0138794.s001" target="_blank">S1 File</a>, for RFP expression). Representative images from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138794#pone.0138794.s007" target="_blank">S6</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138794#pone.0138794.s012" target="_blank">S11</a> Videos are shown. (B) HEK-293 cells stably expressing an IPTG-inducible system for each CPEB knock-down and cells carrying a GFP-3'UTR with mutated CPE (CPE-) were induced or not with IPTG. Two days after induction cells were synchronized by double thymidine blockade (DTB). Samples were collected at the indicated phases of the cell cycle and GFP expression was measured by FACS. Results are shown as the mean value from six experiments, error bars indicate s.d; au, arbitrary units; Sh, short-hairpin; CTRL, control.</p

CPEB1, CPEB2 and CPEB4 are required for cell cycle progression.

<p>(A) HEK-293 cells stably expressing an IPTG-inducible system for CPEB1,2 and 4 knock-down were induced or not with IPTG. Two days after induction cells were synchronized through double thymidine blockade (DTB). Samples were harvested at the indicated time points and protein lysates were analyzed on SDS–PAGE followed by immunoblotting for the indicated proteins. α-tubulin was used as a loading control. (B) HEK-293 cells stably expressing an IPTG-inducible system for each CPEB knock-down were induced or not with IPTG. Three days after induction, cells were marked with EdU for two hours and then released. Samples were harvested at the indicated time points, stained for DNA content with PI and analyzed by FACS. Mean EdU⁺ cell-population: 23100 for control, 10800 for CPEB1-KD cells, 16400 for CPEB2-KD cells, 20900 for CPEB3-KD cells, 18900 for CPEB4-KD cells). Sh, short-hairpin; CTRL, control; IPTG, Isopropyl β-D-1-thiogalactopyranoside; DTB, double-thymidine block; Edu, 5-ethynyl-2'-deoxyuridine; PI, propidium iodide; ES, early S-phase; MS, middle S-phase; LS, late S-phase; FACS, fluorescence-activated cell sorter.</p

Commonly Occurring Cell Subsets in High-Grade Serous Ovarian Tumors Identified by Single-Cell Mass Cytometry.

We have performed an in-depth single-cell phenotypic characterization of high-grade serous ovarian cancer (HGSOC) by multiparametric mass cytometry (CyTOF). Using a CyTOF antibody panel to interrogate features of HGSOC biology, combined with unsupervised computational analysis, we identified noteworthy cell types co-occurring across the tumors. In addition to a dominant cell subset, each tumor harbored rarer cell phenotypes. One such group co-expressed E-cadherin and vimentin (EV), suggesting their potential role in epithelial mesenchymal transition, which was substantiated by pairwise correlation analyses. Furthermore, tumors from patients with poorer outcome had an increased frequency of another rare cell type that co-expressed vimentin, HE4, and cMyc. These poorer-outcome tumors also populated more cell phenotypes, as quantified by Simpson's diversity index. Thus, despite the recognized genomic complexity of the disease, the specific cell phenotypes uncovered here offer a focus for therapeutic intervention and disease monitoring

Commonly Occurring Cell Subsets in High-Grade Serous Ovarian Tumors Identified by Single-Cell Mass Cytometry

Summary: We have performed an in-depth single-cell phenotypic characterization of high-grade serous ovarian cancer (HGSOC) by multiparametric mass cytometry (CyTOF). Using a CyTOF antibody panel to interrogate features of HGSOC biology, combined with unsupervised computational analysis, we identified noteworthy cell types co-occurring across the tumors. In addition to a dominant cell subset, each tumor harbored rarer cell phenotypes. One such group co-expressed E-cadherin and vimentin (EV), suggesting their potential role in epithelial mesenchymal transition, which was substantiated by pairwise correlation analyses. Furthermore, tumors from patients with poorer outcome had an increased frequency of another rare cell type that co-expressed vimentin, HE4, and cMyc. These poorer-outcome tumors also populated more cell phenotypes, as quantified by Simpson’s diversity index. Thus, despite the recognized genomic complexity of the disease, the specific cell phenotypes uncovered here offer a focus for therapeutic intervention and disease monitoring