© 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 1796-1808 doi:10.1242/jcs.198697 RESEARCH ARTICLE Unr defines a novel class of nucleoplasmic reticulum involved in mRNA translation Frédéric Saltel1,2,*,§, Alban Giese1,2,*, Lamia Azzi1,2,3,*, Habiba Elatmani1,2, Pierre Costet4, Zakaria Ezzoukhry1,2,‡, Nathalie Dugot-Senant2, Lucile Miquerol5,Oréda Boussadia6, Harald Wodrich2,7, Pierre Dubus1,2,3,# and Hélenè Jacquemin-Sablon1,2,#,§ ABSTRACT gene expression (Mihailovich et al., 2010; Wolffe, 1994). The Unr (officially known as CSDE1) is a cytoplasmic RNA-binding mammalian Unr proteins, composed of five CSDs, are highly similar protein with roles in the regulation of mRNA stability and translation. In by sharing >90% amino acid identity. Unr is a cytoplasmic RNA- in vitro this study, we identified a novel function for Unr, which acts as a binding protein that, , interacts preferentially with purine-rich positive regulator of placental development. Unr expression studies in motifs located in RNA loops (Jacquemin-Sablon et al., 1994; the developing placenta revealed the presence of Unr-rich foci that Triqueneaux et al., 1999). Unr has been characterized as a regulator of are apparently located in the nuclei of trophoblast giant cells (TGCs). mRNA turnover (Grosset et al., 2000) and translation. During We determined that what we initially thought to be foci, were actually cross translation, Unr acts as a positive or negative regulator of specific sections of a network of double-wall nuclear membrane invaginations transcripts; it either stimulates or represses the translation driven by that contain a cytoplasmic core related to the nucleoplasmic reticulum internal ribosome entry sites (IRESs) (Boussadia et al., 2003; (NR). We named them, accordingly, Unr-NRs. Unr-NRs constitute a Dormoy-Raclet et al., 2005; Hunt et al., 1999; Mitchell et al., 2003) or novel type of NR because they contain high levels of poly(A) RNA and represses cap-dependent translation (Abaza et al., 2006; Duncan et al., translation factors, and are sites of active translation. In murine 2006; Patel et al., 2005). Recent studies have identified numerous Drosophila tissues, Unr-NRs are only found in two polyploid cell types, in TGCs direct Unr mRNA targets in (Mihailovich et al., 2010) and and hepatocytes. In vitro, their formation is linked to stress and in human melanoma (Wurth et al., 2016). In melanoma Unr regulates polyploidy because, in three cancer cell lines, cytotoxic drugs that are its specific target genes mainly at the level of translation elongation or known to promote polyploidization induce their formation. Finally, we termination (Wurth et al., 2016). show that Unr is required in vivo for the formation of Unr-containing Genetic and biochemical studies have linked Unr to several NRs because these structures are absent in Unr-null TGCs. cellular processes as well as to human diseases. Unr has been implicated in the control of cell death (Dormoy-Raclet et al., 2007), KEY WORDS: Unr, CSDE1, Polyploid cells, Nucleoplasmic reticulum, cell differention (Elatmani et al., 2011) and cell migration mRNA translation, Unr knockout (Kobayashi et al., 2013). In humans, recent studies identified important roles for Unr in the promotion of melanoma cell invasion INTRODUCTION and metastasis (Wurth et al., 2016), as well as in disorders, such as Csde1 (cold shock domain-containing E1, also known as and, autism or Diamond-Blackfan anemia (Sanders et al., 2012; Xia hereafterreferredtoasUnr), was identified as a transcription unit et al., 2014; Horos and von Lindern, 2012). Unr has also been located immediately upstream of N-ras in the genome of several characterized as being a critical regulator of embryonic mammalian species (Jeffers et al., 1990; Nicolaiew et al., 1991). development. In Drosophila, Unr inhibits dosage compensation in Unr is a member of the family of proteins that contain an female flies, and Unr overexpression results in predominant male evolutionarily conserved nucleic-acid-binding domain termed cold- lethality at the larvae stage (Patalano et al., 2009). shock domain (CSD). This domain binds single-stranded DNA and Here, we report that Unr is crucial for mouse embryonic and RNA (Graumann and Marahiel, 1998), and CSD-containing proteins placental development. We also identify an Unr-rich structure, are involved in transcriptional and/or post-transcriptional control of forming a network of cytoplasmic invaginations into the nucleus, reminiscent of the nucleoplasmic reticulum (NR) (reviewed in 1INSERM UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, Malhas et al., 2011). These so-called Unr-NRs, found in polyploid F-33000 Bordeaux, France. 2University of Bordeaux, F-33000 Bordeaux, France. cells, constitute a novel type of NR and are found in polyploid cells, 3Department of Tumor Biology, CHU, F-33000 Bordeaux, France. 4Laboratoire de in which active translation takes place. Advance article Transgenese,̀ UniversitéBordeaux, F-33000 Bordeaux, France. 5Aix-Marseille • University, CNRS, IBDM UMR 7288, Marseille, France. 6Taconic Europe, Centre d’Affaires DMCI, Lyon, France. 7MFP CNRS UMR 5234, Microbiologie RESULTS Fondamentale et Pathogénicité, Universitéde Bordeaux, F-33000 Bordeaux, Disruption of the Unr gene in mice causes placental defects France. ‡Present address: UniversitéMohammed 6 des Sciences de la Santé(UM6SS), that coincide with embryonic lethality occurring at mid- Facultéde Médecine, Laboratoire National de Référence (LNR), Casablanca, gestation Morocco. We have previously generated mice carrying an inactivated Unr *,#These authors contributed equally to this work. allele as a result of the deletion of the Unr promoter and reported §Authors for correspondence ([email protected]; helene.jacquemin- that the homozygous mutation of the Unr gene results in a null [email protected]) mutation (Boussadia et al., 2003). The lack of Unr protein led to −/− F.S., 0000-0002-0724-9680 embryonic lethality at mid-gestation, since the percentage of Unr mutant embryos was detected close to the expected Mendelian Received 18 October 2016; Accepted 31 March 2017 levels at 9.5 days of gestation (24%), but were less by day 10.5 Journal of Cell Science 1796 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1796-1808 doi:10.1242/jcs.198697 Fig. 1. Unr disruption causes embryonic and placental abnormal development. (A) Genotype analysis of progeny from Unr+/− intercrosses. The genotypes of the embryos were determined by PCR using DNA extracted from the whole embryo or from the yolk sac, using primers a, b, c and d as shown in Fig. S1B. In parentheses appear the percentages corresponding to the different genotype classes. N.D., genotype not determined. (B) Morphological defects in Unr−/− embryos at E9.5. Micrographs of Unr−/− embryos and control littermates at E9.5 are shown. Note the marked reduction in size of the mutant (right) relative to the control littermate (left) and the absence of neural tube closure (right, the unclosed head is indicated by the arrow). (C) Placental defects in Unr−/− embryos. Histological analysis of placentas of wild-type and Unr KO mice at E8.5, E9.5 and E10.5. Sections through the central regions of placentas were (H&E) stained and imaged at 40×. Maternal deciduas (Ma), Spongiotrophoblast (Sp), trophoblast giant cell (TGC), and Labyrinth (Lab) layers are indicated. (14%) and absent at 12.5 days of gestation and later (Fig. 1A). spongiotrophoblast and labyrinthin layers, and an ∼60−75% decrease Unr−/− embryos were indistinguishable from normal littermates at in the number of TGCs (Fig. 1C). These results show that Unr is E7.5. Between E8.5 and E10.5, mutant embryos could be identified essential for placental development between E8.5 and E11.5. morphologically as they are smaller, present delayed growth and, at E9.5−E10.5, lack neural tube closure (Fig. 1B). Heart maturation Identification of an Unr-rich NR in placental TGCs was delayed in Unr−/−embryos, which presented a defect of We examined the expression of Unr protein within wild-type ventricular trabeculation and smaller than normal atrioventricular placenta at day 10.5. Immunofluorescence analyses of placental cushions at the atrioventricular canal (Fig. S1A). The heart was, sections revealed that Unr is distributed throughout the cytoplasm of Advance article however, not critically abnormal. the three trophoblastic sub-populations (Fig. 2A, left panel). The • Malfunctions of extra-embryonic tissues are primary the cause of specificity of the Unr antibody used in this study was shown by the embryonic lethality at mid-gestation (Copp, 1995; Ihle, 2000; Rossant absence of Unr staining in immunofluorescence analyses of and Cross, 2001). Histological analyses of the yolk sac from Unr−/− placentas of Unr KO mice (Fig. 2B). In addition, we found that mice did not reveal obvious structural changes at E8.5 or later (Fig. Unr also localized to distinguishable foci within the nuclei of a S1B). In contrast, at E9.5−E11.5 obvious placental defects were subset of TGCs, but not of spongiotrophoblasts (Fig. 2A, see detected in placentas of Unr KO mice, when compared to wild-type magnified images of boxed areas). This finding was unexpected littermates. The murine placenta consists of maternal and embryonic because no nuclear expression of Unr has been reported so far, parts, with the latter being composed of three distinct trophoblastic cell prompting us to further characterize these Unr foci through layers; namely, an outermost layer of trophoblast giant cells (TGCs), an physiological, molecular and functional analyses. intermediate spongiotrophoblast layer and the innermost labyrinthin First, we performed, by immunohistochemistry, a temporal layer. In placentas of Unr KO mice there was a marked atrophy of the analysis of the presence of Unr foci in TGCs. The results Journal of Cell Science 1797 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1796-1808 doi:10.1242/jcs.198697 Fig. 2. Unr localizes to discrete foci in nuclei of trophoblast giant cells. (A) Unr immunofluorescence staining of representative sections from wild-type placentas at E10.5.