Department of Biochemistry, Fukuoka Dental College, Fukuoka, Japan; Oral Medicine Research Center, Fukuoka Dental College, Fukuoka, Japan.
Frontier Research Center, Fukuoka Dental College, Fukuoka, Japan.
J Biol Chem. 2020 Aug 21;295(34):12247-12261. doi: 10.1074/jbc.RA119.011870. Epub 2020 Jul 9.
PCBP1, a member of the poly(C)-binding protein (PCBP) family, has the capability of binding heavily oxidized RNA and therefore participates in the cellular response to oxidative conditions, helping to induce apoptosis. There are four other members of this family, PCBP2, PCBP3, PCBP4, and hnRNPK, but it is not known whether they play similar roles. To learn more, we first tested their affinity for an RNA strand carrying two 8-oxoguanine (8-oxoG) residues at sites located in close proximity to each other, representative of a heavily oxidized strand or RNA with one 8-oxoG or none. Among them, only PCBP2 exhibited highly selective binding to RNA carrying two 8-oxoG residues similar to that observed with PCBP1. In contrast, PCBP3, PCBP4, and hnRNPK bound RNA with or without 8-oxoG modifications and exhibited slightly increased binding to the former. Mutations in conserved RNA-binding domains of PCBP2 disrupted the specific interaction with heavily oxidized RNA. We next tested PCBP2 activity in cells. Compared with WT HeLa S3 cells, PCBP2-KO cells established by gene editing exhibited increased apoptosis with increased caspase-3 activity and PARP1 cleavage under oxidative conditions, which were suppressed by the expression of WT PCBP2 but not one of the mutants lacking binding activity. In contrast, PCBP1-KO cells exhibited reduced apoptosis with much less caspase-3 activity and PARP cleavage than WT cells. Our results indicate that PCBP2 as well as PCBP1 bind heavily oxidized RNA; however, the former may counteract PCBP1 to suppress apoptosis under oxidative conditions.
PCBP1 是聚（C）结合蛋白（PCBP）家族的成员之一，具有结合高度氧化 RNA 的能力，因此参与细胞对氧化条件的反应，有助于诱导细胞凋亡。该家族还有其他四个成员，即 PCBP2、PCBP3、PCBP4 和 hnRNPK，但尚不清楚它们是否发挥类似的作用。为了了解更多信息，我们首先测试了它们与一条携带两个 8-氧鸟嘌呤（8-oxoG）残基的 RNA 链的亲和力，这些残基位于彼此非常接近的位置，代表高度氧化的链或带有一个 8-oxoG 或没有 8-oxoG 的 RNA。其中，只有 PCBP2 表现出对携带两个 8-oxoG 残基的 RNA 的高度选择性结合，与 PCBP1 观察到的相似。相比之下，PCBP3、PCBP4 和 hnRNPK 结合带有或不带有 8-oxoG 修饰的 RNA，并且对前者的结合略有增加。PCBP2 保守 RNA 结合结构域的突变破坏了与高度氧化 RNA 的特异性相互作用。接下来，我们在细胞中测试了 PCBP2 的活性。与 WT HeLa S3 细胞相比，通过基因编辑建立的 PCBP2-KO 细胞在氧化条件下表现出更高的细胞凋亡，伴随着 caspase-3 活性的增加和 PARP1 的切割，而 WT PCBP2 的表达可以抑制这种现象，但突变体缺乏结合活性的则不能抑制。相比之下，PCBP1-KO 细胞的细胞凋亡减少，caspase-3 活性和 PARP 切割比 WT 细胞少得多。我们的结果表明，PCBP2 和 PCBP1 都能结合高度氧化的 RNA；然而，前者可能会在氧化条件下与 PCBP1 相互作用以抑制细胞凋亡。
