CPEB and miR-15/16 Co-Regulate Translation of Cyclin E1 mRNA during Xenopus Oocyte Maturation.

Cell cycle transitions spanning meiotic maturation of the Xenopus oocyte and early embryogenesis are tightly regulated at the level of stored inactive maternal mRNA. We investigated here the translational control of cyclin E1, required for metaphase II arrest of the unfertilised egg and the initiation of S phase in the early embryo. We show that the cyclin E1 mRNA is regulated by both cytoplasmic polyadenylation elements (CPEs) and two miR-15/16 target sites within its 3'UTR. Moreover, we provide evidence that maternal miR-15/16 microRNAs co-immunoprecipitate with CPE-binding protein (CPEB), and that CPEB interacts with the RISC component Ago2. Experiments using competitor RNA and mutated cyclin E1 3'UTRs suggest cooperation of the regulatory elements to sustain repression of the cyclin E1 mRNA during early stages of maturation when CPEB becomes limiting and cytoplasmic polyadenylation of repressed mRNAs begins. Importantly, injection of anti-miR-15/16 LNA results in the early polyadenylation of endogenous cyclin E1 mRNA during meiotic maturation, and an acceleration of GVBD, altogether strongly suggesting that the proximal CPEB and miRNP complexes act to mutually stabilise each other. We conclude that miR-15/16 and CPEB co-regulate cyclin E1 mRNA. This is the first demonstration of the co-operation of these two pathways.This study was supported by the Biotechnology and Biological Sciences Research Council (BB/E016316/1). AG was funded by Cancer Research UK and the RATHER consortium, and JA was funded by the Cambridge Overseas Trust and the Parke Davis Bursary (Downing College).This is the final version of the article. It first appeared from PLOS via https://doi.org/10.1371/journal.pone.014679

mRNA spindle localization and mitotic translational regulation by CPEB1 and CPEB4

Transition through cell cycle phases requires temporal and spatial regulation of gene expression to ensure accurate chromosome duplication and segregation. This regulation involves dynamic reprogramming of gene expression at multiple transcriptional and posttranscriptional levels. In transcriptionally silent oocytes, the CPEB-family of RNA-binding proteins coordinates temporal and spatial translation regulation of stored maternal mRNAs to drive meiotic progression. CPEB1 mediates mRNA localization to the meiotic spindle, which is required to ensure proper chromosome segregation. Temporal translational regulation also takes place in mitosis, where a large repertoire of transcripts is activated or repressed in specific cell cycle phases. However, whether control of localized translation at the spindle is required for mitosis is unclear, as mitotic and acentriolar-meiotic spindles are functionally and structurally different. Furthermore, the large differences in scale-ratio between cell volume and spindle size in oocytes compared to somatic mitotic cells may generate distinct requirements for gene expression compartmentalization in meiosis and mitosis. Here we show that mitotic spindles contain CPE-localized mRNAs and translating ribosomes. Moreover, CPEB1 and CPEB4 localize in the spindles and they may function sequentially in promoting mitotic stage transitions and correct chromosome segregation. Thus, CPEB1 and CPEB4 bind to specific spindle-associated transcripts controlling the expression and/or localization of their encoded factors that, respectively, drive metaphase and anaphase/cytokinesis.Fil: Pascual, Rosa. Barcelona Institute Of Science And Technology.; EspañaFil: Segura Morales, Carolina. Barcelona Institute Of Science And Technology.; EspañaFil: Omerzu, Manja. University of Utrecht; Países BajosFil: Bellora, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales. Universidad Nacional del Comahue. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales; ArgentinaFil: Belloc, Eulàlia. Barcelona Institute Of Science And Technology.; EspañaFil: Castellazzi, Chiara Lara. Barcelona Institute Of Science And Technology.; EspañaFil: Reina, Oscar. Barcelona Institute Of Science And Technology.; EspañaFil: Eyras, Eduardo. Universitat Pompeu Fabra; España. Institució Catalana de Recerca i Estudis Avançats; EspañaFil: Maurice, Madelon M.. University of Utrecht; Países BajosFil: Millanes Romero, Alba. Barcelona Institute Of Science And Technology.; EspañaFil: Méndez, Raúl. Barcelona Institute Of Science And Technology.; Españ

CPEB alteration and aberrant transcriptome-polyadenylation lead to a treatable SLC19A3 deficiency in Huntington's disease

Huntington’s disease (HD) is a hereditary neurodegenerative disorder of the basal ganglia for which disease-modifying treatments are not yet available. Although gene-silencing therapies are currently being tested, further molecular mechanisms must be explored to identify druggable targets for HD. Cytoplasmic polyadenylation element binding proteins 1 to 4 (CPEB1 to CPEB4) are RNA binding proteins that repress or activate translation of CPE-containing transcripts by shortening or elongating their poly(A) tail. Here, we found increased CPEB1 and decreased CPEB4 protein in the striatum of patients and mouse models with HD. This correlated with a reprogramming of polyadenylation in 17.3% of the transcriptome, markedly affecting neurodegeneration-associated genes including PSEN1, MAPT, SNCA, LRRK2, PINK1, DJ1, SOD1, TARDBP, FUS, and HTT and suggesting a new molecular mechanism in neurodegenerative disease etiology. We found decreased protein content of top deadenylated transcripts, including striatal atrophy–linked genes not previously related to HD, such as KTN1 and the easily druggable SLC19A3 (the ThTr2 thiamine transporter). Mutations in SLC19A3 cause biotin-thiamine–responsive basal ganglia disease (BTBGD), a striatal disorder that can be treated with a combination of biotin and thiamine. Similar to patients with BTBGD, patients with HD demonstrated decreased thiamine in the cerebrospinal fluid. Furthermore, patients and mice with HD showed decreased striatal concentrations of thiamine pyrophosphate (TPP), the metabolically active form of thiamine. High-dose biotin and thiamine treatment prevented TPP deficiency in HD mice and attenuated the radiological, neuropathological, and motor HD-like phenotypes, revealing an easily implementable therapy that might benefit patients with HD

Identification of a new deadenylation negative feedback loop that regulates meiotic progression

Els oòcits de vertebrats es troben aturats a la profase I de la primera meiosi (PI). Durant el procés anomenat oogènesi, els oòctits sintetitzen i emmagatzemen grans quantitats d'ARN missatgers(ARNm)que els seran necessaris per la compleció de la meiosi.I,per posteriorment, aturar-se de nou a la metafase de la segona divisió meiòtica (MII) per l'activitat del factor citostàtic(CSF).D'aquestes divisions en destaca el fet que transcorren en absència de transcripció, i per tant depenen totalment en l'activació traduccional dels ARNm anteriorment esmentats que han estat acumulats durant l'oogènesi. L'activació traduccional d'aquests missatgers és principalment induïda per l'elongació de les cues d'adenines(cues de poli(A)), aquest procés és mediat per les seqüències de poliadenilació citoplasmàtiques (CPE)presents a la regió 3' no tradudïda (3'UTR)dels ARNm. El moment i la longitud de la poliadenilació dels ARNm que contenen CPEs estan finament regulats, de manera que en combinació amb la degradació de proteïnes, s'estableixen els patrons específics d'expresió de les proteïnes que condueixen la meiosi (Shmitt et al., 2002; de Moor and Richter, 1997; Ballantyne et al., 1997; Mendez et al., 2002; Charlesworth et al., 2002). Fins a la data, no s'havia descrit que la deadenilació (escurçament de la cua de poli(A)) fos necessària per la progressió meiòtica. En aquesta tesi s'ha descrit, a partir d'un cribatge d'abast genòmic, una ruta de retroalimentació negativa requerida per a la sortida de la primera metafase meiòtica. La nova ruta identificada, a més té la particularitat d'actuar a nivell traduccional regulant l'expressió de proteïnes que participen directament en la progressió meiòtica. L'element central d'aquesta nova ruta és la proteïna C3H-4, que a la vegada és regulada per poliadenilació citoplasmàtica. C3H-4 crea la retroalimentació negativa interaccionant amb elements ARE de les regions 3'UTR, promovent la deadenilació del ARNm al qual s'uneix. D'entre les seves dianes hem identificat Emi1 i Emi2, ambdós reguladors de l'activitat de l'APC/C, crítica per la divisió cel·lular

A fly trap mechanism provides sequence-specific RNA recognition by CPEB proteins

Cytoplasmic changes in polyA tail length is a key mechanism of translational control and is implicated in germline development, synaptic plasticity, cellular proliferation, senescence, and cancer progression. The presence of a U-rich cytoplasmic polyadenylation element (CPE) in the 3′ untranslated regions (UTRs) of the responding mRNAs gives them the selectivity to be regulated by the CPE-binding (CPEB) family of proteins, which recognizes RNA via the tandem RNA recognition motifs (RRMs). Here we report the solution structures of the tandem RRMs of two human paralogs (CPEB1 and CPEB4) in their free and RNA-bound states. The structures reveal an unprecedented arrangement of RRMs in the free state that undergo an original closure motion upon RNA binding that ensures high fidelity. Structural and functional characterization of the ZZ domain (zinc-binding domain) of CPEB1 suggests a role in both protein–protein and protein–RNA interactions. Together with functional studies, the structures reveal how RNA binding by CPEB proteins leads to an optimal positioning of the N-terminal and ZZ domains at the 3′ UTR, which favors the nucleation of the functional ribonucleoprotein complexes for translation regulation.ISSN:0890-9369ISSN:1549-547

The miRISC as well as the putative miR-15/16 target sites in the cyclin E1 3’UTR are functional.

<p><b>A.</b> Tethering of either Ago2 or the effector domain of GW182 represses translation of a reporter in mammalian cells as well as in <i>Xenopus</i> oocytes. Schematic representation of reporters used. mRNAs encoding lambda-N peptide with an HA-tag or an HA-tag only, fused to GW182 effector domain or Ago2 were either injected into oocytes for 24 h prior to injection of <i>Renilla</i> luciferase reporter mRNA containing 3’UTR Box B sites (Rluc-BoxB) or transfected into HeLa cells 24 h prior to transfection of plasmids encoding the reporter genes. An mRNA/plasmid encoding Firefly luciferase was co-injected/co-transfected as an internal control. After a 6 h incubation, cells were harvested and reporter protein expression was assessed. The experiment was repeated 3 times with similar results. <b>B</b>. Repression of a reporter mRNA tethered to the effector domain of GW182 is independent of a poly(A) tail. Schematic representation of reporters used. The experiment was performed in <i>Xenopus</i> oocytes as in A, using either non-adenylated (pA-) or <i>in vitro</i> polyadenylated (pA+) RNA. RNA was assessed from a pool of 50 injected oocytes. <b>C.</b> The two miR-15/16 sites co-operate in repressing translation of the cyclin E1 3’UTR. Luciferase reporters containing the wild-type cyclin E1 3’UTR (WT), mutations in the first miR target site (miR mut1), the second target site (miR mut2) or both (miR mut) were injected into stage VI oocytes. <i>Renilla</i> luciferase mRNA was co-injected as an internal control. Firefly luciferase levels are expressed as a ratio to <i>Renilla</i> internal control. The graph displays the results for a representative experiment. <b>D</b>. The miR-15/16 target sites are active in a degradation-independent manner. Luciferase reporters containing the wild-type cyclin E1 3’UTR (WT) or the cyclin E1 miR mut 3’UTR were injected into oocytes (see C for further details), in the presence of co-injected control or miR-15/16 LNAs, as indicated. * Student t-test P<0.01. RNA extracted from injected oocytes was subjected to reverse transcription and quantitative real-time PCR to assess RNA levels, which are expressed as a ratio of firefly to <i>Renilla</i> luciferase. The graph displays the results for a representative experiment.</p

CPEB complexes and miRISC cooperate in the regulation of translational activation of cyclin E1 during oocyte maturation.

<p><b>A.</b> Components of the RISC complex can associate with the translational silencing complex in immature oocytes (-p). Oocytes were injected with HA-Ago2 mRNA, and the resulting lysates were subjected to immunoprecipitation with anti-HA antibodies. Input represents 5% of the immunoprecipitated fractions. Western blotting was performed with anti-HA or -CPEB1 antibodies and visualised by ECL.–p–no progesterone, immature oocytes; +p–oocytes matured with progesterone. <b>B</b>. miRNAs co-immunoprecipitate with CPEB complexes. Lysates from uninjected oocytes or oocytes expressing a FLAG-tagged Xp54 were used for immunoprecipitation using a monoclonal anti-CPEB antibody, an isotype-matched control (His), or in the case of FLAG-Xp54 expressing oocytes and uninjected control (“-“), an anti-FLAG antibody (FLAG). RNA was isolated and real-time RT-PCR performed for the miRNAs and small RNAs indicated. Relative quantities were normalised per oocyte input. For plotting purposes all enrichment values were scaled to the enrichment of miR-15 in the CPEB immunoprecipitate of Experiment 2.</p

Inhibition of miR-15/16 causes premature polyadenylation of cyclin E1 mRNA and acceleration of meiotic maturation.

<p><b>A.</b> Injection of a molar excess of CPE-containing RNA competitor enhances the effect of miR site mutations. 500 fmol of either a control 85 nt RNA not containing any CPE sequences (no CPE UTR) or a 65 nt 3’–terminal sequence of the cyclin B1 3’UTR (cyclin B1 UTR) were injected into stage VI oocytes. After an overnight incubation, oocytes were re-injected with either the WT or miR mut Firefly reporter constructs and the <i>Renilla</i> control RNA. The oocytes were lysed and assayed for luciferase after 6 h. The graph represents and average of 3 experiments with the WT reporter normalised to 1 in each case. (** P<0.01, 2-tailed paired t-test). <b>B</b>. Uninjected stage VI oocytes (u.i) or oocytes injected with control LNA (ctrl LNA) or a mixture of anti-miR-15 and anti-miR-16 LNAs (15+16 LNA) were incubated overnight, following which they were stimulated with progesterone. Samples were taken at indicated times. Extracted RNA was subjected to RNA-ligation coupled PCR polyadenylation analysis using primers for indicated mRNAs. Vertical red line indicates early polyadenylation in 15+16 LNA injected sample. Panel shows a representative experiment of 4. Graph underneath cyclin E panel depicts ImageJ profile plot taken along the dotted line. <b>C</b>. 100–150 stage VI oocytes were injected with either control LNA (ctrl LNA) or anti-miR-15 and anti-miR-16 LNAs (15+16 LNA) and incubated overnight and then treated with progesterone. GVBD was scored by the appearance of a white spot on the animal pole of the oocyte. The time required for 50% of the oocytes to achieve GVBD (GVBD-50) is plotted for 5 independent experiments.</p

Mature forms of miR-15 and miR-16 are present in <i>Xenopus</i> oocytes.

<p><b>A</b>. Alignment of putative miR-15/16 seed binding sites across vertebrate cyclin E1 3’UTRs. <b>B</b>. Alignment of vertebrate miR-15b and miR-16a reveals nearly perfect conservation of the two miRNAs, with the seed sequence highlighted in bold. <b>C</b>. miR-15b and 16a levels do not undergo significant changes during oogenesis and oocyte maturation. Levels of the two microRNAs were verified by qPCR alongside the control U2 snRNA. The graph represents absolute quantities throughout oogenesis in three independent biological samples.</p

Translation of cyclin E1 during oocyte maturation is CPE-dependent.

<p><b>A.</b> Cyclin E1 mRNA is present throughout oogenesis. RT-qPCR was carried out on total RNA extracted from staged oocytes and eggs. The graph represents the relative amount of cyclin E1 mRNA compared to GAPDH mRNA, with values of each transcript set to 1 for stage I. Representative of 5 progesterone maturation experiments. <b>B</b>. Cyclin E1 protein is not expressed until late in oocyte maturation following the polyadenylation of its mRNA. Stage VI oocytes were incubated with progesterone for the indicated times and the corresponding lysates were analysed by Western blot using cyclin E1 and CPEB antibodies (upper panels). RNA extracted from these oocytes was subjected to RNA-ligation-coupled PCR poly(A) analysis (lower panel). *—non-specific band. <b>C.</b> The cyclin E1 3’UTR contains a cluster of three CPE sequences and an additional one overlapping the hexanucleotide as well as two putative miR-15/16 target sites as indicated. Not to scale. <b>D</b>. CPE sequences repress translation in immature oocytes, and activate translation in eggs. Schematic representation of reporters used. Firefly luciferase reporters containing a control 3’UTR (luc400), the wild-type cyclin E1 3’UTR (WT) or the 3’UTR with mutations in the CPEs (CPE mut) were injected into stage VI oocytes. <i>Renilla</i> luciferase reporter mRNA was co-injected as an internal control. Oocytes were incubated for 24h with or without progesterone and both sets were assayed for luciferase expression. Firefly luciferase levels are expressed as a ratio to <i>Renilla</i> internal control. <b>E</b>. CPE sequences in the cyclin E1 3’UTR direct polyadenylation during oocyte maturation. Radiolabelled RNAs representing the WT 3’-terminal 180 nt of the cyclin E1 3’UTR and the same fragment with mutated CPE sequences were injected into oocytes, and maturation was induced by progesterone. RNA extracted from untreated oocytes and progesterone-matured eggs was analysed by denaturing gel electrophoresis and autoradiography.</p