K259-SUMOylation of DGCR8 promoted by p14ARF exerts a tumor-suppressive function.
K259-SUMOylation of DGCR8 promoted by p14ARF exerts a tumor-suppressive function.
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p14ARF 促进的 DGCR8 K-259-SUMO 化发挥肿瘤抑制功能
DOI：
10.1093/jmcb/mjw030
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发表时间：
2016-10
期刊：
影响因子：
--
通讯作者：
Yu J
中科院分区：
文献类型：
--
作者：
Zhu C;Chen C;Chen R;Deng R;Zhao X;Zhang H;Duo J;Chen Q;Jin H;Wang Y;Huang J;Xu M;Yu J
亲爱的编辑，DGCR 8对于miRNA生物发生是必不可少的（Han et al.，2004，2006; Quick-Cleveland等人，2014年）。我们最近报道了DGCR 8在K707的SUMO化影响其与siRNA的亲和力，并控制pri-miRNAs在基因沉默中的直接功能。然而，K707 R突变并不去除DGCR 8的所有SUMO 1修饰（Zhu等人，2015），表明DGCR 8具有其他SUMO化位点。在这里，我们确定了K259作为DGCR 8 SUMO化的替代位点，其直接由细胞核中的p14 ARF促进。我们还证明了K259-SUMO化在阻止DGCR 8核输出中的显著作用，这是其在将pri-miRNAs加工成pre-miRNAs和肿瘤抑制中的功能所必需的。为了研究p14 ARF是否与DGCR 8结合以促进其SUMO化，进行Ni 2+-NTA pull-down测定。p14 ARF（WT）显著增强DGCR 8的SUMO化，而截短形式的p14 ARF △△则没有（图1A和B）。据报道，p14 ARF可以通过与UBC 9的结合来增强其结合配偶体的SUMO化（Rizos等人，2005年）。实际上，我们观察到外源性p14 ARF和内源性DGCR 8之间的相互作用（补充图S1 A和B）。免疫荧光试验还显示，p14 ARF，而不是p14 ARF △△，与DGCR 8共定位于核仁中（补充图S1 C）。我们进一步研究了p14 ARF是否与内源性Drosha结合，Drosha是微处理器复合体中DGCR 8的重要伙伴。结果显示p14 ARF和Drosha之间没有相互作用（补充图S1 D和E）。这些数据表明p14 ARF直接与DGCR 8相互作用，促进其SUMO化。DGCR 8可以在K 707处SUMO化（Zhu等人，2015年）。然而，突变体DGCR 8 K707 R与p14 ARF的相互作用不受影响（补充图S2 A），p14 ARF仍然增强其SUMO化（补充图S2 B），这与我们先前的观察一致，即K707是DGCR 8的主要但不是唯一的SUMO化位点。为了鉴定DGCR 8的p14 ARF结合结构域，产生了一系列具有相当表达水平的截短DGCR 8形式，包括DG 1 -275（1-275 aa）、DG 1 -483、DG 1 -614和DG 276 -773（补充图S2 C）。Co-IP结果显示，p14 ARF与DG 1 -275、DG 1 -483和DG 1 -614强烈相互作用，但不与DG 276 -773相互作用（补充图S2 D），这表明结合主要通过由DGCR 8的核定位信号（NLS）组成的DG 1 -275进行。此外，所有含有N-末端区域（1-275 aa）的截短DGCR 8形式均为SUMO化（补充图S2 E）。这些数据表明p14 ARF可能促进DGCR 8 N端区域（1-275 aa）的SUMO化。事实上，在与p14 ARF共表达的情况下，DG 1 -275的SUMO化水平显著增强，而DG 276 -773的SUMO化水平没有显著增强当DG 1 -275进一步分段为DG 1 -140和DG 141 -275时，只有DG 141 -275的SUMO化水平可以被p14 ARF增强。（补充图S3 A），表明另一个SUMO化位点位于DGCR 8的N-末端区域（141-275 aa）内。因此，我们通过K222 R的点突变或K259/260 R的双突变产生突变的DGCR 8，并在100 μ mol/L条件下检测它们的SUMO化水平。
Dear Editor, DGCR8 is essential for the miRNA biogenesis (Han et al., 2004, 2006; Quick-Cleveland et al., 2014). We recently reported that at K 707-SUMOylation of DGCR8 influences its affinity with primiRNAs and controls the direct functions of pri-miRNAs in gene silencing. However, the K707R mutation does not remove all SUMO1 modifications of DGCR8 (Zhu et al., 2015), suggesting that DGCR8 bears other SUMOylation sites. Here, we identified K 259 as an alternative site for DGCR8 SUMOylation that was directly promoted by p14ARF in the nucleus. We also demonstrated a notable role of K 259-SUMOylation in preventing DGCR8 nuclear export that is required for its function in the processing of pri-miRNAs into pre-miRNAs and tumor suppression. To investigate whether p14ARF associates with DGCR8 to promote its SUMOylation, the Ni 2+-NTA pull-down assay was performed. p14ARF (WT) significantly enhanced DGCR8 SUMOylation whereas the truncated form p14ARF△△ did not (Figure 1 A and B). It was reported that p14ARF can enhance SUMOylation of its binding partners through the association with UBC9 (Rizos et al., 2005). Indeed, we observed mutual interactions between exogenous p14ARF and endogenous DGCR8(Supplementary Figure S1A and B). The immunofluorescence assay also showed that p14ARF, but not p14ARF△△, co-localized with DGCR8 in the nucleolus (Supplementary Figure S1C). We further examined whether p14ARF binds to endogenous Drosha, an important partner of DGCR8 in microprocessor complex. The results showed no interaction between p14ARF and Drosha (Supplementary Figure S1D and E). These data indicated that p14ARF directly interacted with DGCR8 to promote its SUMOylation. DGCR8 can be SUMOylated at K 707 (Zhu et al., 2015). However, the mutant DGCR8 K707R was not affected for its interaction with p14ARF (Supplementary Figure S2A) and its SUMOylation was still enhanced by p14ARF (Supplementary Figure S2B), which was consistent with our previous observation that K 707 is a major but not the only SUMOylation site of DGCR8. To identify the p14ARF-binding domain (s) of DGCR8, a series of truncated DGCR8 forms with comparable expression levels were generated, including DG1-275 (1–275 aa), DG1-483, DG1-614, and DG276-773 (Supplementary Figure S2C). Co-IP results showed that p14ARF strongly interacted with DG1-275, DG1-483, and DG1-614 but not DG276-773 (Supplementary Figure S2D), suggesting that the binding was mainly through DG1-275 composed of a nuclear localization signal (NLS) of DGCR8. In addition, all truncated DGCR8 forms containing the N-terminal region (1–275 aa) were SUMOylated (Supplementary Figure S2E). These data suggested that SUMOylation on the N-terminal region (1–275 aa) of DGCR8 may be promoted by p14ARF. Indeed, the SUMOylation level of DG1-275 but not DG276-773 was significantly enhanced under the co-expression with p14ARF (Figure 1 C).When DG1-275 was further segmented into DG1-140 and DG141-275, only the SUMOylation level of DG141-275 can be enhanced by p14ARF (Supplementary Figure S3A), suggesting that another SUMOylation site was located within the N-terminal region (141–275 aa) of DGCR8. Therefore, we generated mutated DGCR8 by point-mutation of K222R or doublemutation of K259/260R and examined their SUMOylation levels under the
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