Abstract：
Cytoplasmic polyadenylation element-binding protein 4 (Cpeb4) is an RNA-binding protein that regulates posttranscriptional regulation, such as regulation of messenger RNA stability and translation. In the previous study, we reported that Cpeb4 localizes to nuclear bodies upon induction of osteoclast differentiation by RANKL. However, the mechanisms of the localization of Cpeb4 and osteoclastogenesis by Cpeb4 remain unknown. Here, we show that Cpeb4 localizes to the nuclear bodies by its RNA-binding ability and partially regulates normal splicing during osteoclast differentiation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with Phos-tag® revealed that the phosphorylation levels of Cpeb4 were already high in the RAW264.7 cells and were not altered by RANKL treatment. Immunofluorescence showed that exogenous Cpeb4 in HEK293T cells without RANKL stimulation localized to the same foci as shown in RANKL-stimulated RAW264.7 cells. Furthermore, when nuclear export was inhibited by leptomycin B treatment, Cpeb4 accumulated throughout the nucleus. Importantly, RNA recognition motif (RRM) 7 of Cpeb4 was essential for the localization. In contrast, the intrinsically disordered region, RRM1, and zinc finger domain CEBP_ZZ were not necessary for the localization. The mechanistic study showed that Cpeb4 co-localized and interacted with the splicing factors serine/arginine-rich splicing factor 5 (SRSF5) and SRSF6, suggesting that Cpeb4 may be involved in the splicing reaction. RNA-sequencing analysis revealed that the expression of genes related to cell proliferation processes, such as mitotic cell cycle and regulation of cell cycle processes, was elevated in osteoclasts depleted of Cpeb4. Interestingly, the splicing pattern of the inhibitor of DNA binding 2 (Id2) gene, which suppresses osteoclast differentiation, was altered by the depletion of Cpeb4. These results provide new insight into the role of Cpeb4 as a player of normal splicing of Id2 in osteoclast differentiation.
摘要：
细胞质聚腺苷酸化元件结合蛋白4（Cpeb4）是一种RNA结合蛋白，可调节转录后调控，如调节信使RNA的稳定性和翻译。在之前的研究中,我们报道，在RANKL诱导破骨细胞分化后，Cpeb4定位于核体。然而,Cpeb4的定位和Cpeb4的破骨细胞生成的机制仍然未知。这里,我们表明，Cpeb4通过其RNA结合能力定位到核体，并在破骨细胞分化过程中部分调节正常剪接。使用Phos-tag®的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示,在RAW264.7细胞中,Cpeb4的磷酸化水平已经很高,并且没有被RANKL处理改变。免疫荧光显示,没有RANKL刺激的HEK293T细胞中的外源Cpeb4定位在与RANKL刺激的RAW264.7细胞中所示相同的病灶。此外，当核出口被乐霉素B治疗抑制时，Cpeb4在整个细胞核中积累。重要的是,Cpeb4的RNA识别基序(RRM)7对于定位是必需的。相比之下,内在无序的区域，RRM1和锌指结构域CEBP_ZZ不是定位所必需的。机制研究表明，Cpeb4与剪接因子丝氨酸/富含精氨酸的剪接因子5（SRSF5）和SRSF6共定位并相互作用，提示Cpeb4可能参与剪接反应。RNA测序分析显示，与细胞增殖过程相关的基因表达，如有丝分裂细胞周期和细胞周期过程的调节，在消耗了Cpeb4的破骨细胞中升高。有趣的是,DNA结合抑制剂2（Id2）基因的剪接模式，抑制破骨细胞分化，因Cpeb4的耗尽而改变。这些结果为Cpeb4作为Id2正常剪接者在破骨细胞分化中的作用提供了新的见解。
