Laboratory of Signaling in Biomolecular Systems, Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil.
Laboratory of Genome Instability, Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil.
Biol Cell. 2024 May;116(5):e2300128. doi: 10.1111/boc.202300128. Epub 2024 Mar 27.
The dual-specificity phosphatase 3 (DUSP3) regulates cell cycle progression, proliferation, senescence, and DNA repair pathways under genotoxic stress. This phosphatase interacts with HNRNPC protein suggesting an involvement in the regulation of HNRNPC-ribonucleoprotein complex stability. In this work, we investigate the impact of DUSP3 depletion on functions of HNRNPC aiming to suggest new roles for this enzyme.
The DUSP3 knockdown results in the tyrosine hyperphosphorylation state of HNRNPC increasing its RNA binding ability. HNRNPC is present in the cytoplasm where it interacts with IRES trans-acting factors (ITAF) complex, which recruits the 40S ribosome on mRNA during protein synthesis, thus facilitating the translation of mRNAs containing IRES sequence in response to specific stimuli. In accordance with that, we found that DUSP3 is present in the 40S, monosomes and polysomes interacting with HNRNPC, just like other previously identified DUSP3 substrates/interacting partners such as PABP and NCL proteins. By downregulating DUSP3, Tyr-phosphorylated HNRNPC preferentially binds to IRES-containing mRNAs within ITAF complexes preferentially in synchronized or stressed cells, as evidenced by the higher levels of proteins such as c-MYC and XIAP, but not their mRNAs such as measured by qPCR. Under DUSP3 absence, this increased phosphorylated-HNRNPC/RNA interaction reduces HNRNPC-p53 binding in presence of RNAs releasing p53 for specialized cellular responses. Similarly, to HNRNPC, PABP physically interacts with DUSP3 in an RNA-dependent manner.
Overall, DUSP3 can modulate cellular responses to genotoxic stimuli at the translational level by maintaining the stability of HNRNPC-ITAF complexes and regulating the intensity and specificity of RNA interactions with RRM-domain proteins.
双特异性磷酸酶 3（DUSP3）在遗传毒性应激下调节细胞周期进程、增殖、衰老和 DNA 修复途径。这种磷酸酶与 HNRNPC 蛋白相互作用，表明其参与调节 HNRNPC-核糖核蛋白复合物的稳定性。在这项工作中，我们研究了 DUSP3 耗竭对 HNRNPC 功能的影响，旨在为这种酶提出新的作用。
DUSP3 的敲低导致 HNRNPC 的酪氨酸过度磷酸化状态，增加其 RNA 结合能力。HNRNPC 存在于细胞质中，在那里它与 IRES 反式作用因子（ITAF）复合物相互作用，该复合物在蛋白质合成过程中招募 40S 核糖体到 mRNA 上，从而促进含有 IRES 序列的 mRNA 的翻译，以响应特定刺激。与此一致，我们发现 DUSP3 存在于 40S、单体和多聚体中，与 HNRNPC 相互作用，就像其他先前鉴定的 DUSP3 底物/相互作用伙伴（如 PABP 和 NCL 蛋白）一样。通过下调 DUSP3，Tyr 磷酸化的 HNRNPC 优先与 ITAF 复合物中的包含 IRES 的 mRNA 结合，这在同步化或应激细胞中更为明显，如 c-MYC 和 XIAP 等蛋白质水平升高，但 qPCR 测量的其 mRNA 水平没有升高。在 DUSP3 缺失的情况下，这种增加的磷酸化-HNRNPC/RNA 相互作用减少了 RNA 释放的 p53 与 HNRNPC 的结合，从而使 p53 能够对特定的细胞反应做出反应。同样，与 HNRNPC 一样，PABP 以 RNA 依赖的方式与 DUSP3 物理相互作用。
总之，DUSP3 可以通过维持 HNRNPC-ITAF 复合物的稳定性并调节 RRM 结构域蛋白与 RNA 的相互作用的强度和特异性，在翻译水平上调节细胞对遗传毒性刺激的反应。
