Department of Molecular Pharmacology, Faculty of Pharmaceutical Science, Graduate School of Pharmaceutical Sciences, Tokyo University of Science, Noda, Chiba, Japan.
J Cell Physiol. 2024 Apr;239(4):e31197. doi: 10.1002/jcp.31197. Epub 2024 Jan 29.
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.
细胞质多聚腺苷酸化元件结合蛋白 4（Cpeb4）是一种 RNA 结合蛋白，可调节信使 RNA 的稳定性和翻译等转录后调控。在之前的研究中，我们报道了 Cpeb4 在 RANKL 诱导破骨细胞分化时定位于核体内。然而，Cpeb4 的定位机制和 Cpeb4 对破骨细胞分化的影响仍不清楚。在这里，我们显示 Cpeb4 通过其 RNA 结合能力定位于核体内，并在破骨细胞分化过程中部分调节正常剪接。SDS-聚丙烯酰胺凝胶电泳分析与 Phos-tag®揭示 RAW264.7 细胞中的 Cpeb4 磷酸化水平已经很高，且不受 RANKL 处理的影响。免疫荧光显示，在没有 RANKL 刺激的情况下，外源性 Cpeb4 在 HEK293T 细胞中与 RANKL 刺激的 RAW264.7 细胞中显示的相同焦点定位。此外，当用莱普霉素 B 处理抑制核输出时，Cpeb4 积累在整个细胞核中。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，无序区 RRM1 和锌指结构域 CEBP_ZZ 对于定位不是必需的。机制研究表明，Cpeb4 与剪接因子丝氨酸/精氨酸丰富剪接因子 5（SRSF5）和 SRSF6 共定位并相互作用，表明 Cpeb4 可能参与剪接反应。RNA 测序分析显示，Cpeb4 缺失的破骨细胞中与细胞增殖过程相关的基因，如有丝分裂细胞周期和细胞周期过程的调节，表达上调。有趣的是，Id2 基因（抑制破骨细胞分化）的剪接模式因 Cpeb4 的缺失而改变。这些结果为 Cpeb4 作为破骨细胞分化中 Id2 正常剪接的参与者提供了新的见解。
