The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation.
The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation.
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DOI：
10.1126/science.abc9359
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发表时间：
2020-12-18
期刊：
影响因子：
--
通讯作者：
Bartel DP
中科院分区：
文献类型：
--
作者：
Shi CY;Kingston ER;Kleaveland B;Lin DH;Stubna MW;Bartel DP
microRNAs（miRNAs）是一种在人类和其他动物中广泛存在的基因抑制的短RNA。每个miRNA与Argonaute（AGO）蛋白结合以形成沉默复合物，其中miRNA与靶mRNA内的位点配对，并且AGO募集引起靶转录物的不稳定或翻译减少的机制。大多数miRNA是相当长的寿命，因为与AGO的关联保护它们免受细胞核酸酶的侵害。然而，一些miRNAs相对不稳定。这些miRNA的不稳定性的一个潜在解释来自于观察到与某些靶位点的相互作用可以促进miRNA的破坏，从而颠倒了典型的调控逻辑。这种靶向miRNA降解（target-directed miRNA degradation，TDMD）现象被一些病毒利用，这些病毒产生转录物，这些转录物触发特定宿主miRNA的衰变，否则这些特定宿主miRNA将阻碍病毒复制。此外，最近发现四种内源性转录物触发TDMD。这些包括CYRANO非编码RNA，其指导miR-7的有效降解。然而，这种现象可能加速其他miRNAs降解的程度尚不清楚，部分原因是尚未鉴定出TDMD所需的蛋白质。触发TDMD的不寻常靶位点与介导基因阻遏的典型位点不同，因为它们不仅与miRNA的5′区域配对，而且还与miRNA的3′区域广泛配对。这种与3′区域的配对可以引起构象变化，使miRNA 3′末端暴露于添加或去除核苷酸的酶-分别称为加尾和修剪的过程。在存在触发TDMD的位点的情况下观察到的拖尾和修剪已被认为是TDMD途径的专性步骤。然而，负责靶向拖尾的酶的损失对TDMD的影响很小，这表明TDMD可能通过另一种机制发生。为了更多地了解这种机制，我们进行了CRISPRi筛选，旨在鉴定CYRANO介导的miR-7降解所需的蛋白质。我们的筛选揭示了CYRANO指导的miR-7降解以及其他已知的TDMD实例所需的TDM 8。此外，在各种哺乳动物细胞以及果蝇S2细胞和线虫中，在敲除TDMWIM 8直系同源物后增加的miRNA的鉴定涉及数十种额外的miRNA作为内源性TDMD的底物。事实上，对于已知miRNA半衰期的细胞，TDMD解释了大多数短寿命miRNA的不稳定性。TDM 8是Cullin-RING E3泛素连接酶的底物受体，这表明TDMD的替代模型不依赖于miRNA加尾和修剪。在该模型中，EMWIM 8泛素连接酶识别与miRNA 3′区域广泛配对后发生的构象变化，这导致AGO的多聚泛素化。然后，多泛素化的AGO被26 S蛋白酶体降解，从而使miRNA暴露于细胞质核酸酶。支持这一模型，抑制泛素化或蛋白酶体介导的蛋白水解阻碍TDMD。此外，作为TDMD底物的miRNAs在EMWIM 8的存在下优先与多泛素化蛋白结合，并且AGO表面上的赖氨酸取代废除了TDMD，暗示AGO是泛素连接酶的直接靶标。TDMD需要TDMWIM 8泛素连接酶，支持TDMD模型，其中AGO的靶向蛋白水解使miRNA暴露于降解。此外，TDMD的隐含范围远远超出了先前在哺乳动物和其他脊椎动物中确定的四个内源性例子。内源性TDMD似乎影响了哺乳动物细胞中的数十种miRNA，并且它延伸到果蝇和线虫，这意味着它自两侧动物的最后共同祖先以来一直在塑造miRNA水平和动态。miRNAs对靶标作用的分子模型，反之亦然。在miRNA介导的调节过程中（上图），miRNA将AGO引导至mRNA，mRNA募集缩短mRNA poly（A）尾的蛋白质，导致更快的mRNA降解。在TDMD过程中（下图），与miRNA 3′区域广泛配对的靶位点诱导构象变化，这些变化被TDMWIM 8泛素连接酶识别。这种连接酶使AGO聚泛素化，引起其蛋白水解，这使miRNA暴露于降解。ncRNA，非编码RNA。microRNA（miRNAs）与Argonaute（AGO）蛋白结合，指导广泛的转录后基因抑制。尽管与AGO的结合通常可以保护miRNA免受核酸酶的攻击，但与一些不寻常的靶RNA的广泛配对可以触发miRNA降解。在这里，我们发现这种靶向的miRNA降解（TDMD）需要Cullin-RING E3泛素连接酶。这一发现和其他研究结果表明并支持TDMD的机制模型，其中AGO通过泛素-蛋白酶体途径的靶向蛋白水解暴露miRNA进行降解。此外，功能丧失研究表明，Cullin-RING连接酶可加速哺乳动物、苍蝇和线虫细胞中大量miRNA的降解，从而确定大多数短寿命miRNA的半衰期。这些结果阐明了TDMD的机制，并扩展了其在双侧性动物中形成miRNA水平的推断作用。一些microRNA靶点触发Argonaute蛋白的蛋白水解，为塑造microRNA水平提供了广泛使用的机制。
MicroRNAs (miRNAs) are short RNAs that direct widespread gene repression in humans and other animals. Each miRNA associates with an Argonaute (AGO) protein to form a silencing complex in which the miRNA pairs to sites within target mRNAs, and AGO recruits machinery that causes destabilization or reduced translation of the targeted transcript. Most miRNAs are quite long-lived because association with AGO protects them from cellular nucleases. However, some miRNAs are relatively unstable. A potential explanation for the instability of these miRNAs comes from the observation that interactions with certain target sites can promote miRNA destruction, thereby inverting the typical regulatory logic. This phenomenon of target-directed miRNA degradation (TDMD) is exploited by some viruses that produce transcripts that trigger the decay of specific host miRNAs that would otherwise impede viral replication. Moreover, four endogenous transcripts have recently been found to trigger TDMD. These include the CYRANO noncoding RNA, which directs efficient degradation of miR-7. However, the extent to which this phenomenon might accelerate degradation of other miRNAs has been unclear—in part because proteins required for TDMD had not been identified. The unusual target sites that trigger TDMD differ from the typical sites that mediate gene repression in that they not only pair to the 5′ region of the miRNA but also extensively pair to the miRNA 3′ region. This pairing to the 3′ region can cause conformational changes that expose the miRNA 3′ terminus to enzymes that append or remove nucleotides—processes called tailing and trimming, respectively. The tailing and the trimming observed in the presence of sites that trigger TDMD have been proposed to be obligate steps of the TDMD pathway. However, loss of an enzyme responsible for target-directed tailing has little influence on TDMD, suggesting that TDMD might occur through another mechanism. To learn more about this mechanism, we carried out a CRISPRi screen designed to identify proteins required for CYRANO-directed degradation of miR-7. Our screen revealed that ZSWIM8 is required for CYRANO-directed miR-7 degradation, as well as for other known examples of TDMD. Moreover, identification of miRNAs that increased after knocking out the ZSWIM8 ortholog in various mammalian cells, as well as in Drosophila S2 cells and nematodes, implicated dozens of additional miRNAs as substrates of endogenous TDMD. Indeed, for cells in which miRNA half-lives were known, TDMD explained the destabilization of most short-lived miRNAs. ZSWIM8 is the substrate receptor of a Cullin-RING E3 ubiquitin ligase, suggesting an alternative model for TDMD that does not depend on miRNA tailing and trimming. In this model, the ZSWIM8 ubiquitin ligase recognizes the conformational changes that occur upon extensive pairing to the miRNA 3′ region, which leads to polyubiquitination of AGO. Polyubiquitinated AGO is then degraded by the 26S proteasome, thereby exposing the miRNA to cytoplasmic nucleases. Supporting this model, inhibiting of either ubiquitination or proteasome-mediated proteolysis impeded TDMD. Moreover, the miRNAs that were TDMD substrates preferentially associated with polyubiquitinated proteins in the presence of ZSWIM8, and substitutions of lysines on the surface of AGO abrogated TDMD, implicating AGO as the direct target of the ubiquitin ligase. TDMD requires the ZSWIM8 ubiquitin ligase, supporting a model of TDMD in which target-directed proteolysis of AGO exposes the miRNA for degradation. Furthermore, the implied scope of TDMD extends far beyond the four endogenous examples previously identified in mammals and other vertebrates. Endogenous TDMD appears to influence dozens of miRNAs in mammalian cells, and it extends to Drosophila and nematodes, implying that it has been shaping miRNA levels and dynamics since the last common ancestor of bilaterian animals. Molecular models for effects of miRNAs on targets, and vice versa. During miRNA-mediated regulation (top), the miRNA directs AGO to an mRNA, which recruits proteins that shorten the mRNA poly(A) tail, causing more rapid mRNA degradation. During TDMD (bottom), a target site with extensive pairing to the miRNA 3′ region induces conformational changes that are recognized by the ZSWIM8 ubiquitin ligase. This ligase polyubiquitinates AGO, causing its proteolysis, which exposes the miRNA for degradation. ncRNA, noncoding RNA. MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to direct widespread post-transcriptional gene repression. Although association with AGO typically protects miRNAs from nucleases, extensive pairing to some unusual target RNAs can trigger miRNA degradation. Here we found that this target-directed miRNA degradation (TDMD) required the ZSWIM8 Cullin-RING E3 ubiquitin ligase. This and other findings suggested and supported a mechanistic model of TDMD in which target-directed proteolysis of AGO by the ubiquitin–proteasome pathway exposes the miRNA for degradation. Moreover, loss-of-function studies indicated that the ZSWIM8 Cullin-RING ligase accelerates degradation of numerous miRNAs in cells of mammals, flies, and nematodes, thereby specifying the half-lives of most short-lived miRNAs. These results elucidate the mechanism of TDMD and expand its inferred role in shaping miRNA levels in bilaterian animals. Some microRNA targets trigger proteolysis of Argonaute proteins, providing a widely employed mechanism for sculpting microRNA levels.
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影响因子：
64.5
作者：
Kleaveland B;Shi CY;Stefano J;Bartel DP
通讯作者：
Bartel DP
影响因子：
14.9
作者：
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通讯作者：
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82.9
作者：
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通讯作者：
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影响因子：
7
作者：
Kingston, Elena R.;Bartel, David P.
通讯作者：
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影响因子：
9.2
作者：
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通讯作者：
Brown, Brian D.