The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation

doi:10.1126/science.abc9359

GSTDTAP > 气候变化

DOI	10.1126/science.abc9359
	The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation
	Charlie Y. Shi; Elena R. Kingston; Benjamin Kleaveland; Daniel H. Lin; Michael W. Stubna; David P. Bartel
	2020-12-18
发表期刊	Science
出版年	2020
英文摘要	MicroRNAs help to regulate many genes in animal cells. Each microRNA associates with an Argonaute (AGO) protein, forming a complex in which the microRNA pairs with a messenger RNA (mRNA) target and AGO recruits factors that accelerate mRNA decay. However, for some unusual targets, the reverse occurs: Pairing to the target recruits factors that accelerate microRNA decay rather than degradation of the mRNA. Working independently, Shi et al. and Han et al. elucidate the mechanism of this phenomenon. They found that pairing to the unusual targets recruits a ubiquitin ligase that causes degradation of AGO, thereby exposing the microRNA to cellular nucleases. Mutating the ubiquitin ligase in diverse animals and cell types deregulates numerous microRNAs, implying that this phenomenon is widely deployed to sculpt microRNA levels. Science , this issue p. [eabc9359][1], p. [eabc9546][2] ### INTRODUCTION MicroRNAs (miRNAs) are short RNAs that direct widespread gene repression in the cells of 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. In the aggregate, miRNAs reduce the output of most human genes, and most of the 90 broadly conserved miRNA families found in mammals are required for viability or proper development. 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, which produce transcripts that trigger the decay of specific host miRNAs that would otherwise impede viral replication. In addition, four endogenous transcripts have recently been found to trigger TDMD. These include the CYRANO long noncoding RNA, which directs efficient degradation of miR-7. However, the extent to which this phenomenon might accelerate the degradation of other miRNAs has been unclear, in part because proteins required for TDMD had not been identified. ### RATIONALE 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 pair extensively to the miRNA 3′ region. This extensive 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 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 CYRANO-directed degradation of miR-7, which suggests 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 in cultured human cells. ### RESULTS Our screen revealed that the ZSWIM8 protein was required for CYRANO-directed miR-7 degradation. ZSWIM8 was also required for other known examples of TDMD, including degradation triggered by a viral noncoding RNA and a cellular mRNA. Moreover, identification of miRNAs that increased after knocking out ZSWIM8 in different types of mouse and human cells 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. Similar experiments that examined the consequences of knocking out the ZSWIM8 ortholog in Drosophila cells and nematodes indicated that endogenous TDMD also shapes miRNA levels and dynamics in invertebrate species. ZSWIM8 is the substrate receptor of a Cullin-RING E3 ubiquitin ligase; this suggests 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 26 S proteasome, thereby exposing the miRNA to cytoplasmic nucleases. In support of this model, knockdown of other known components of the ZSWIM8 ubiquitin ligase impeded TDMD, as did inhibition of ubiquitin activation, of general Cullin-RING ligase activity, or of proteasome-mediated proteolysis. 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, which implicates AGO as the direct target of the ubiquitin ligase. ### CONCLUSION The finding that TDMD requires the ZSWIM8 ubiquitin ligase supports 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, which suggests that it has been shaping miRNA levels and dynamics since the last common ancestor of bilaterian animals. ![Figure][3] 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 posttranscriptional gene repression. Although association with AGO typically protects miRNAs from nucleases, extensive pairing to some unusual target RNAs can trigger miRNA degradation. We found that this target-directed miRNA degradation (TDMD) required the ZSWIM8 Cullin-RING E3 ubiquitin ligase. This and other findings support 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. [1]: /lookup/doi/10.1126/science.abc9359 [2]: /lookup/doi/10.1126/science.abc9546 [3]: pending:yes
领域	气候变化 ; 资源环境
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文献类型	期刊论文
条目标识符	http://119.78.100.173/C666/handle/2XK7JSWQ/308359
专题	气候变化资源环境科学
推荐引用方式 GB/T 7714	Charlie Y. Shi,Elena R. Kingston,Benjamin Kleaveland,et al. The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation[J]. Science,2020.
APA	Charlie Y. Shi,Elena R. Kingston,Benjamin Kleaveland,Daniel H. Lin,Michael W. Stubna,&David P. Bartel.(2020).The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation.Science.
MLA	Charlie Y. Shi,et al."The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation".Science (2020).