YB-1 Is Important for an Early Stage Embryonic Development

The eukaryotic Y-box-binding protein-1 (YB-1) is involved in the transcriptional and translational control of many biological processes, including cell proliferation. In clinical studies, the cellular level of YB-1 closely correlates with tumor growth and prognosis. To understand the role of YB-1 in vivo, especially in the developmental process, we generated YB-1 knock-out mice, which are embryonic lethal and exhibit exencephaly associated with abnormal patterns of cell proliferation within the neuroepithelium. β-Actin expression and F-actin formation were reduced in the YB-1 null embryo and YB-1-/- mouse embryonic fibroblasts, suggesting that the neural tube defect is caused by abnormal cell morphology and actin assembly within the neuroepithelium. Fibroblasts derived from YB-1-/- embryos demonstrated reduced growth and cell density. A colony formation assay showed that YB-1-/- mouse embryonic fibroblasts failed to undergo morphological transformation and remained contact-inhibited in culture. These results demonstrate that YB-1 is involved in early mouse development, including neural tube closure and cell proliferation.

The Y-box protein family is characterized by a highly conserved cold-shock domain that binds nucleic acids and shares homology with the prokaryotic cold-shock proteins (1,2). The human Y-boxbinding gene, YB-1, is located on chromosome 1p34 (1). YB-1 has multiple functions but was initially identified as a transcription factor that associates with the Y-box sequence of the major histocompatibility complex class II genes (3).
YB-1 promotes cell proliferation through its transcriptional regulation of target genes such as proliferating cell nuclear anti-gen (PCNA), 2 epidermal growth factor receptor, DNA topoisomerase II␣, thymidine kinase, and DNA polymerase ␣ (4, 5). We previously reported its role in the transcriptional activation of human multidrug resistance 1 (MDR1) and DNA topoisomerase II␣ in response to various environmental stimuli (6,7). In addition, it has been shown to chaperone RNA, modify chromatin, participate in the translational masking of mRNA, and be involved in stress responses such as the redox signaling pathway (8). Eukaryotic Y-box proteins also regulate gene expression at the translational level through their recognition of RNA (9 -11), and therefore play critical roles in both mRNA turnover and translational control.
YB-1 protects mammalian cells from the cytotoxic effects induced by DNA damage. We previously reported that human cancer cell lines overexpressing YB-1 resist cisplatin, whereas the reduction of YB-1 itself leads to increased sensitivity to cisplatin, other DNA-interacting drugs, and UV irradiation (2). YB-1 is mainly localized in the cytoplasm, but translocates to the nucleus following UV irradiation of cells or treatment with anticancer drugs (12). YB-1 binds directly to repair-associated proteins such as PCNA and p53 (13), whereas proteolytic cleavage of the C-terminal fragment is linked to stress induced by DNA damage (14).
In clinical studies, the cellular level of YB-1 has been shown to correlate with tumor growth and prognosis in cancers of the ovary, lung, and breast (15). Moreover, overexpression or the nuclear presence or absence of YB-1 plays a critical role in P-glycoprotein expression, malignant progression, poor prognosis, and global drug resistance (2,15,16).
To understand how YB-1 proteins exert their multiple functions, we previously established mouse embryonic stem cell lines with a heterozygously targeted disruption of the YB-1 gene (YB-1 ϩ/Ϫ ), and we demonstrated their hypersensitivity to cytotoxic agents such as cisplatin and mitomycin C (17).
Here we carried out targeted disruptions of the mouse YB-1 gene to elucidate the role of YB-1 molecules in vivo. We show that YB-1 plays a critical role in early development in mice. The targeted disruptions were fatal in the late embryonic stage, and animals showed defects in the anterior neural tube. Furthermore, we investigated the role of YB-1 in cell proliferation and the transformation activity of MEFs.

EXPERIMENTAL PROCEDURES
Animals-Animals were mated overnight, and the females were examined for a vaginal plug the following morning. Noon on the day of vaginal plug detection was recorded as day E0.5. All animal experiments were carried out according to the guidelines for animal experimentation at Kyushu University, Japan, and the University of Occupational Environmental Health, Japan. All experimental protocols were approved by the ethics committee of Kyushu University and the University of Occupational Environmental Health, Japan.
In Situ Hybridization-In situ hybridization of digoxygeninlabeled probes was performed as described previously (18). The digoxigenin-labeled hybridization probe was prepared from an in vitro transcription system (Promega, Madison, WI) using the mouse YB-1 full-length cDNA (11).
Immunohistochemistry-Cells seeded the previous day on glass coverslips were washed with phosphate-buffered saline (PBS), fixed with 3.7% formaldehyde for 30 min, rinsed twice with PBS, and then incubated with PBS containing 0.1% Triton X-100 (Sigma) for 30 min. Next, the coverslips were washed with PBS, incubated with 10% goat serum for 1 h at room temperature in a humidified container, and then incubated for 1 h with FITC-conjugated phalloidin (Sigma). After washing three times with PBS, glass slides were mounted using Slowfade mounting medium (Molecular Probes). FITC-conjugated phalloidin (Sigma) was diluted 1:200 and used to detect F-actin organization in mouse tissue and MEF cells.
Immunohistochemical Analysis of Mouse Embryo Sections-Mouse embryo tissue was fixed with 10% buffered formalin and embedded in paraffin. Sagittal sections (5 m thick) were cut and mounted on silane-coated glass slides. After routine deparaffination and rehydration through gradient ethanol immersions, the slides were steam-heated for 20 min to expose the antigen. Endogenous peroxidase activity was quenched using 3% (v/v) H 2 O 2 followed by three 5-min washes in PBS containing 0.2% (v/v) Triton X-100, and the sections were blocked with 10% (v/v) normal goat serum in PBS. Specimens were incubated for 1 h with the YB-1 and ␤-actin antibody diluted in PBS containing 0.3% (v/v) Triton X-100 and 0.1% (w/v) bovine serum albumin, followed by three 5-min washes in PBS, and then incubation with the FITC-conjugated goat anti-rabbit antibody (Kirkegaard & Perry Laboratories, Gaithersburg, MD) for 30 min. Specimens were counterstained with hematoxylin for 30 s and washed with tap water. The sections were immediately dehydrated by sequential immersion in gradient ethanol and xylene, then mounted with Permount (ProSciTech, Australia), and coverslips. Images were obtained using a Leica DMRX upright microscope coupled with a digital camera (Leica, Germany).

deficiency causes embryonic lethality
Embryos at the indicated stages were isolated from intercrosses of heterozygous animals, and the total numbers (n) of intact as well as disintegrated or resorbed embryos were counted. For calculation of % expected (Ϫ/Ϫ): n(Ϫ/Ϫ) ϫ 100 ϫ 3/n(ϩ/ϩ) ϩ n(ϩ/Ϫ). serum) conditions. Dishes were trypsinized and counted daily using a Coulter-type cell size analyzer (CDA-500, Sysmex, Kobe, Japan). Transformation Assay-Cells (3 ϫ 10 3 ) were seeded in triplicate in 10-cm dishes and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotic-antimycotic (Invitrogen). Growth medium was changed every 3 days. After 14 -16 days, transformation efficiency was evaluated by counting individual foci. All transformation assays were repeated at least three times. Representative plates were stained with Giemsa and photographed.

Stage
Anchorage-independent Growth-Growth in soft agar was assayed in 35-mm dishes prepared with a lower layer of 0.7% agar (Invitrogen) overlaid with top agar (0.4%) containing 5 ϫ 10 3 suspended cells. Cells were fed every 3 days with media. Fifteen days after plating, colonies were stained with 2% crystal violet, and colonies with Ͼ50 cells were counted on an inverted microscope (Olympus, Tokyo, Japan).
Knockdown Analysis Using siRNA-siRNA transfections were performed according to the manufacturer's instructions (Invitrogen). Briefly, cells cultured in 35-mm dishes were transfected with stealth RNA interference-negative control duplexes and YB-1 siRNA oligonucleotides (CAACGUCG-GUAUCGCCGAAACUUCA) at a final concentration of 100 M using Lipofectamine 2000 (Invitrogen). After transfection, cell number and cell volume were quantified using an electronic sizing technique with a CDA-500 Coulter-type cell size analyzer (Sysmex). Cells were also harvested for Western blotting (17).

Disruption of YB-1 Causes Embryonic
Lethality-To elucidate the function of YB-1 during mouse development, we used gene targeting to generate YB-1-deficient mice. Although the heterozygous offspring appeared normal and fertile (Table 1), Southern blot analysis of tail DNA from 3-week-old mice revealed that no live animals (of 144 births from heterozygote crosses) were homozygous for the YB-1 mutation. Thus, loss of YB-1 results in embryonic lethality.
To determine the time at which the YB-1 mutant becomes lethal, we examined embryos from YB-1 ϩ/Ϫ intercrosses at various developmental stages. PCR genotyping data of nine mouse embryos at E11.5 revealed two wild-types, five heterozygotes, and two homozygous mutants, in accordance with the expected Mendelian ratio ( Fig. 1A and confirmed by PCR with four additional primer sets; data not shown). In contrast to wild-type embryos, the growth of YB-1 Ϫ/Ϫ embryos appeared retarded as early as E10.5 (Fig. 1B). Most YB-1 Ϫ/Ϫ embryos had been resorbed by E17.5 and YB-1 Ϫ/Ϫ embryos died between E14.5 and E18.5 ( Table 1). The phenotype of YB-1 Ϫ/Ϫ embryos includes retarded growth, hemorrhage, and severe anemia but is otherwise normal in appearance (Fig. 1C). Mouse YB-1 Is Expressed in Most Tissues during Embryogenesis-We reported previously that human YB-1 is expressed ubiquitously in the adult (19). The YB-1 transcript and protein have also been detected in mouse embryonic stem cells (17). To determine whether the expression of mouse YB-1 is developmentally regulated, we performed in situ hybridization on mouse embryos tissue sections at E13.5. We found that mouse YB-1 mRNA is expressed at whole body, specifically at high levels in the brain region (Fig. 2, A and B). Expression in the brain is widespread, with some enrichment in the cortical plate, diencephalons (thalamus), roof of the neopallial cortex, and choroid plexus extending into lateral ventricle, midbrain, and cerebellar primordium (Fig. 2B). YB-1 mRNA is also strongly expressed in the posterior mesoderm, the craniofacial region, root ganglion, kidney, liver, head mesoderm, and in the developing heart (Fig. 2B). These data support a critical role for YB-1 expression during embryonic development.
YB-1 protein expression in E13.5 embryos is almost ubiquitous, with high expression detected in the central nervous system, lung, kidney, and heart (Fig. 2C). YB-1 was predominantly localized to the cytoplasm region in wild-type embryos. No expression was detected in either connective tissues or bone of wild-type embryos and was absent from YB-1 Ϫ/Ϫ embryos (Fig. 2C).
Neural Tube Closure Is Impaired in YB-1-deficient Mice-As shown in Fig. 1B, YB-1 Ϫ/Ϫ embryos were smaller than their wild-type littermates, although no gross abnormalities were observed in organ or limb development. When examined at E10.5 to E13.5, ϳ30% of the YB-1 Ϫ/Ϫ embryos exhibited exencephaly in the forebrain, midbrain, and hindbrain regions (12/80 YB-1 Ϫ/Ϫ embryos) (Fig. 1B). Almost all of the mutant embryos were pale and anemic, as a consequence of severe blood loss through hemorrhage (seen as petechial and paintbrush patterns in Fig. 1C).
Histological analysis of other parts of the mutant embryos revealed that the YB-1 mutation does not affect organogenesis, because all major organs were intact (data not shown). Exencephaly typically reflects a defect in closure of the anterior neu-  DECEMBER 29, 2006 • VOLUME 281 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 40443 ral tube, which normally occurs between E8.5 and E9.5 (20). Fig.  3 showed a severe brain malformation characterized by exencephaly, expanded midbrain, and a disrupted cortical zone. Examination of older embryos (E13.5 to E15.5) revealed that the mutant brains failed to develop further.

Embryonic Lethality of YB-1-deficient Mice
Frontal and cross-sections of the hindbrain region of the E11.5 to E13.5 neural tube defect (NTD) embryos clearly demonstrate incomplete neural tube closure (Fig. 3, B and C), and the anterior neural tubes of most E10.5 to E11.5 YB-1 Ϫ/Ϫ embryos failed to close with varying degrees of severity. No other cranial or neural tube abnormalities such as holoprosencephaly or impaired caudal neural closure were observed. Those YB-1 Ϫ/Ϫ mice that achieved skull closure also possessed major brain structures but demonstrated retarded development of the maxilla and mandible (Fig. 3D). Most E15.5 YB-1 Ϫ/Ϫ embryos had a subcutaneous edema of the whole body (Fig. 3D), which was not observed in wild-type and heterozygous mice. Moreover, fetal livers of YB-1 Ϫ/Ϫ embryos were smaller than those of their YB-1 ϩ/Ϫ and YB-1 ϩ/ϩ littermates, which is suggestive of hepatic hematopoiesis (Fig. 3E). YB-1 Ϫ/Ϫ embryos were also anemic as a result of macroscopically detectable defects in erythropoiesis of the fetal livers (Fig. 3E). These data suggest that exencephaly, smaller size of organ, and severe hemorrhage account for the embryonic lethality of the YB-1 mutation.
During the first three passages, cell proliferation and population doubling was comparable between YB-1 ϩ/ϩ , YB-1 ϩ/Ϫ , and YB-1 Ϫ/Ϫ MEFs. From passages 4 to 5 onward, all YB-1 Ϫ/Ϫ MEFs analyzed showed greatly reduced proliferation and a reduction in cell numbers under base-line culture conditions (Fig. 4C). YB-1 ϩ/Ϫ and YB-1 ϩ/ϩ MEFs proliferated at a similar rate. YB-1 Ϫ/Ϫ MEFs exhibited premature senescence and an extended crisis as determined by an enlarged and flattened cell morphology (Fig. 5B). After 100 days of culture, YB-1 Ϫ/Ϫ MEF cells showed reduced cell proliferation and density, which could be completely recovered to wild-type levels by expression of the YB-1 vector (Fig. 4D). YB-1 expression from this vector was confirmed by Western blotting (Fig. 4B). These data demonstrate the importance of YB-1 in cell proliferation and maintaining cell morphology.
NTD and Actin Assembly-NTDs involving mutations in genes that regulate actin arrangement at the cell membrane or play alternative roles in actin synthesis have been reported previously (21). In all cases, the defects included exencephaly caused by a failure of cranial neural fold elevation, as observed in the YB-1 Ϫ/Ϫ embryos. In addition, YB-1 has been shown to associate with ␤-actin mRNA and the actin protein (11,22). We used immunofluorescence to investigate whether ␤-actin synthesis and rearrangement are affected in E13.5 YB-1 Ϫ/Ϫ embryos, and we showed that ␤-actin protein levels were greatly reduced in the cephalic region of the YB-1 null embryo, in comparison with the wild type (Fig. 5A).
Phalloidin staining of E13.5 brain sections revealed a substantially decreased accumulation of F-actin along the basal edge of neuroepithelial cells in the null mutant embryo compared with the wild type (Fig. 5B). These data suggest that the reduced ␤-actin levels and F-actin filament formation might be responsible for the NTDs of YB-1 Ϫ/Ϫ embryos. In some mutant animals, a reduced apical constriction of the neuroepithelial cells within this region was also observed.
We next examined the role of YB-1 in cell morphology and organization of the actin cytoskeleton. Wild-type MEFs had an elongated morphology and an F-actin-rich polarized cytoskeleton. In contrast, YB-1 Ϫ/Ϫ MEFs were round in shape, with lower cell density (Fig. 5B). Most strikingly, mutant cells lacked appreciable F-actin structures such as fibers or bundles. Instead, a small amount of F-actin was seen as a fuzzy phalloidin signal that was consistently found in the subcellular region rather than in the cell perimeter (Fig. 5B). These results show that YB-1 is essential for organizing F-actin and maintaining the cell shape of MEFs.
As YB-1 possesses RNA binding activity and has been shown to regulate protein synthesis and mRNA stability (11,23), we next investigated the interaction of YB-1 with ␤-actin mRNA.  DECEMBER 29, 2006 • VOLUME 281 • NUMBER 52

Embryonic Lethality of YB-1-deficient Mice
An in vitro RNA gel shift assay was performed using purified recombinant YB-1 and a probe corresponding to full-length ␤-actin mRNA. Recombinant YB-1 clearly bound to ␤-actin mRNA, whereas the control glutathione S-transferase protein failed to do so (supplemental Fig. 1A). To determine whether the interaction occurs in vivo, we performed reverse transcrip-  A, at low magnification, ␤-actin was shown to be ubiquitously expressed in wild-type embryos; however, YB-1 Ϫ/Ϫ embryos demonstrated local reduction and derangement of ␤-actin expression. This is especially obvious in connective tissue-filled central nervous system supportive tissues. B, immunohistochemistry with FITC-phalloidin was performed in E10.5 mouse embryos (top panels) and MEFs (bottom panels). Mutant embryos showed reduced F-actin structures. Normal cytoskeletal structures can be seen in wild-type MEFs. Stress fiber formation was reduced in YB-1 Ϫ/Ϫ MEFs, and cells were flatter and larger than wild-type MEFs.
tion-PCR using ␤-actin-specific primers on mRNA isolated by co-immunoprecipitation with YB-1. ␤-Actin transcript was amplified from wild-type but not from YB-1 Ϫ/Ϫ MEFs (supplemental Fig. 1B), suggesting that YB-1 indeed interacts with ␤-actin mRNA in MEFs. This interaction might regulate the activity or availability of ␤-actin in protein synthesis.
Reduced Anchorage-independent Growth by YB-1 Ϫ/Ϫ Cells-We established three wild-type and three YB-1 Ϫ/Ϫ immortalized MEF lines after continuous culturing for more than 6 months to investigate their spontaneous transformation ability in vitro. Although the wild-type cells did not show any signs of a decrease in proliferative rate, YB-1 Ϫ/Ϫ MEFs failed to undergo morphological transformation and remained contactinhibited after 2 weeks of cultivation (Fig. 6A, upper and middle  panel). Following re-expression of transgenic YB-1, however, the MEFs underwent morphological transformation, whereas vector-only transduced MEFs failed to do so (Fig.   6A, lower panel). Furthermore, a one-fifth reduction in anchorage-independent growth was observed in the YB-1 Ϫ/Ϫ MEF clones (Fig. 6B).
To confirm these results, we investigated whether knockdown of endogenous YB-1 via siRNA affected cell growth and size. The siRNA oligonucleotide was directed against the YB-1 C-terminal region, with the exception of the cold-shock domain. Western blot analysis of siRNA-transfected MEFs revealed that YB-1 protein levels were reduced to 20% of wildtype levels 72 h after transfection (Fig. 7A). YB-1 siRNA-transfected MEFs also showed a reduced growth rate and were ϳ10% larger (22 m in diameter) than the negative control transfected MEFs (20 m in diameter) (Fig. 7, B and C). This phenomenon was consistent with our earlier observations of YB-1 Ϫ/Ϫ MEFs (Fig. 5B) and shows that YB-1 is involved in both regulating cell growth rates and cell size. In an anchorageindependent transformation assay in soft agar, YB-1 expressing MEFs (number 70) showed morphological transformation, but siRNA-transfected MEFs demonstrated reduced transformation activity (Fig. 7D). These results confirm our earlier finding that YB-1 is necessary for anchorage-independent transformation activity (see also Fig. 6A).

DISCUSSION
This study demonstrates that YB-1 plays a critical role in DNA repair, transcription, mRNA turnover, and translational control. Previously, Lu et al. (24) reported that targeted disruption of YB-1 exon 3 (encoding part of the cold-shock domain) causes embryonic lethality and showed that YB-1 is important for cellular stress responses and prevention of premature senescence after E13.5. In this study, we demonstrated that YB-1 Ϫ/Ϫ embryos exhibit severe growth retardation and progressive mortality after E10.5, revealing a nonredundant role for YB-1 in early embryonic development. Our study design disrupted YB-1 exons 5 and 6, encoding a nonspecific RNA binding region of the protein. Western blot analysis using an antibody against the YB-1 C terminus revealed that the YB-1 protein was completely absent from the E13.5 YB-1 Ϫ/Ϫ embryo (Fig. 4A).
In this experiment, we first demonstrated that ␤-actin expression and F-actin formation were reduced in the YB-1 null embryo and YB-1 Ϫ/Ϫ MEF, suggesting that the neural tube defect is caused by abnormal cell morphology and actin assembly within the neuroepithelium. We also showed that YB-1 Ϫ/Ϫ MEFs failed to undergo morphological transformation in culture cells and suggested that YB-1 is involved in cell proliferation.
Although only 20% of YB-1 null mutant mice showed exencephaly (Table 1), this is not an unusual finding, as mouse embryos subjected to inactivation of a critical gene via homologous recombination rarely show NTDs with complete penetrance (25). As an NTD phenotype, exencephaly reflects the failure of neural fold elevation in well defined, mechanistically distinct elevation zones (26). The genes mutated in several mouse NTD models that are involved in actin regulation (Abl/ Arg, Marcs, Mena/Profilin1, Mlp, Sprm, Vcl) support the postulated role for actin in neural fold elevation and suggest that the NTDs are caused by an absence of the morphogenetic force normally provided by the apical redistribution of actin (25). We observed that YB-1 impairs translation of the ␤-actin transcript in a rabbit reticulocyte translation system (data not shown). Similar results have been reported for ␤-actin (27) and ␣-globin mRNAs (28). The strong, nonspecific in vitro binding of YB-1 to mRNA inhibits translation (29) and is a possible mechanism for regulating actin activity or its availability in protein synthesis. This is consistent with our finding that disruption of YB-1 leads to low ␤-actin levels and reduced actin assembly (Fig. 5).
Recently, the localization of ␤-actin mRNA to sites of active actin polymerization has been shown to modulate cell migration and neurite outgrowth (30). This localization requires the oncofetal protein Zipcode-binding protein 1 (ZBP1), which promotes translocation of the ␤-actin transcript to actin-rich protrusions in primary fibroblasts and neurons. ZBP1 associates with the ␤-actin transcript in the nucleus and prevents premature translation in the cytoplasm by blocking translation initiation. Interestingly, Matsumoto et al. (27) reported an interaction between YB-1 and ZBP1, suggesting that both pro-teins might coordinate in their regulation of ␤-actin mRNA localization, protection, and protein synthesis at the correct site. Further elucidation of this interaction should improve the understanding of the molecular mechanisms behind ␤-actin regulation.
The role of YB-1 in cell proliferation might be executed through its interaction with actin (22), as actin filaments form the contractile ring that cleaves the cell during cytokinesis (31). Alternatively, cell proliferation might be regulated by the effect of YB-1 on the cell cycle proteins cyclin A and cyclin B1, as YB-1 was found to induce strongly elevated levels of cyclin B1 protein in the mitotic stage (32). The targeted disruption of one allele of the chicken Y-box protein gene in DT40 cells results in major defects in the cell cycle (33). In this study, no difference in cyclin A and cyclin B expression was observed in YB-1 Ϫ/Ϫ mouse embryos or MEFs (data not shown), suggesting that the expression level of these proteins did not cause the embryonic lethality and abnormality of the YB-1 Ϫ/Ϫ mice.
Bergmann et al. (34) showed that transgenic mice expressing human hemagglutinin-tagged YB-1 developed diverse breast carcinomas through the induction of genetic instability caused by mitotic failure and centrosome amplification. We observed the spontaneous transformation of wild-type MEFs but showed that YB-1 Ϫ/Ϫ MEFs failed to undergo morphological transformation and remained contact-inhibited (Fig. 6B). Re-expression of YB-1 restored the transformation activity suggesting that YB-1 is necessary for tumor promotion. Indeed, overexpression of YB-1 mRNA and protein is a hallmark of several human malignant diseases (2,34), whereas the level of YB-1 protein expression has been linked with the prognosis of breast cancer patients and resistance to chemotherapeutic agents (5).
The nuclear translocation of YB-1 requires phosphorylation by the signal transduction protein Akt (35), which plays a role in tumor formation and progression. Evdokimova et al. (36) reported that phosphorylation by Akt also regulates the association of YB-1 with the capped 5Ј terminus of mRNA and that activated Akt might relieve translational repression of YB-1-bound mRNA. We investigated the level of Akt protein in wild-type and siRNA-mediated YB-1 knockdown MEFs, but no difference was detected (Figs. 4A and 7A), suggesting that YB-1 does not affect Akt protein levels in MEFs.
Target of rapamycin is a downstream kinase in the PI3K/ Akt signaling pathway that phosphorylates S6K and translation initiation factor 4E-binding protein (4EBP), thus regulating translation (37). We also observed that S6K protein levels were reduced in YB-1 null mouse embryos, suggesting that YB-1 might be involved in this PI3K signaling pathway. Indeed, YB-1 is transcriptionally down-regulated in PI3Ktransformed and Akt-transformed cells (29,38). YB-1 acts downstream of the target of rapamycin, as the phosphorylation levels of S6K and 4EBP are unchanged in YB-1-overexpressing cells (39). An independent line of evidence has revealed the essential role of protein synthesis in PI3K-and Akt-induced transformation (40).
Activation of eukaryotic elongation factor 1A (eEF-1A) through phosphorylation by S6K (41,42) enables it to bind to actin and regulate its activity or its availability in protein synthesis (43,44). eEF-1A mutants have severe defects in cell morphology, the actin cytoskeleton, and actin bundling (44). In mammalian systems, disruption of the actin cytoskeleton results in reduced translation. In this study, we observed that YB-1 co-precipitated with eEF-1A (supplemental Fig. S2), suggesting that eEF-1A might compensate for the function of YB-1 in YB-1 Ϫ/Ϫ embryos and MEFs. We also observed that another translational regulatory protein, EF-1, was overexpressed in YB-1 Ϫ/Ϫ E11.5 embryos and siRNA-mediated YB-1 knockdown MEFs (Fig. 4A and 7A), indicative of an alternative compensatory mechanism.
In conclusion, we have described the function of YB-1 in the mouse embryo and in MEFs. We show that it is involved in mouse embryo development, neural tube defects, and cell proliferation.