The Ccr4-Not complex monitors the translating ribosome for codon optimality.
The Ccr4-Not complex monitors the translating ribosome for codon optimality.
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DOI：
10.1126/science.aay6912
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发表时间：
2020-04-17
期刊：
影响因子：
--
通讯作者：
Beckmann R
中科院分区：
文献类型：
--
作者：
Buschauer R;Matsuo Y;Sugiyama T;Chen YH;Alhusaini N;Sweet T;Ikeuchi K;Cheng J;Matsuki Y;Nobuta R;Gilmozzi A;Berninghausen O;Tesina P;Becker T;Coller J;Inada T;Beckmann R
严格控制的基因表达过程需要信使rna (mrna)，它代表dna衍生的多肽蓝图，由细胞的蛋白质生产机器核糖体翻译。因此，蛋白质水平在很大程度上取决于细胞mRNA水平，而mRNA衰减的控制是设定基因表达总体水平的最关键过程之一。mRNA的半衰期在不同转录本之间差异很大，mRNA衰变速率的调节与mRNA翻译的延伸期密切相关。为此，密码子最优性已被确立为确定多种真核生物mRNA半衰期的关键参数。也已经确定，富含非最佳密码子的短寿命mrna的及时衰变需要Ccr4-Not复合体。Ccr4-Not是一种必需的蛋白质复合物，在mRNA降解中发挥着最重要的作用，它作为主要的细胞质3 ' -poly(A)-tail deadenylase，启动大多数mRNA的衰变。通过死基化和随后的mRNA脱冠机制的激活，Ccr4-Not复合体使mRNA能够被主要的降解外切酶（如位于5 ‘端的Xrn1和位于3 ’端的外切体）所接触。通过Ccr4-Not复合物监测和协调mRNA衰变的密码子最优性的分子机制仍然难以捉摸。由于非最优密码子影响核糖体的解码动力学，mRNA降解主要发生在共译过程中，因此密码子最优性是直接在核糖体上监测的，这是非常可信的。此外，参与的Ccr4-Not复合物与核糖体之间存在直接的物理联系，并且该复合物的Not4亚基（E3连接酶）使酵母40S核糖体亚基的eS7蛋白泛素化。因此，我们开始通过结合冷冻电子显微镜（cro - em）、核糖体分析和生化分析来深入了解mRNA稳态背景下Ccr4-Not复合物与翻译机制之间的联系。我们使用来自酿酒酵母的亲和纯化的天然Ccr4-Not -核糖体复合物进行低温电镜分析，发现Ccr4-Not通过Not5亚基募集到核糖体上。Not5的N端，特别是一个3 α-螺旋束，与核糖体e位点特异性相互作用，Not5 N端的缺失导致Ccr4-Not复合物失去稳定的核糖体结合。然而，小核糖体亚基蛋白eS7通过Not4亚基的泛素化仍然发生。除了转移RNA （tRNA）和核糖体RNA （RNAs）外，Not5相互作用还涉及小亚基的核糖体蛋白eS25。我们发现Ccr4-Not与起始核糖体和延长核糖体相互作用。在这两种情况下，Not5仅在核糖体采用一种独特的构象时才与e位点结合，在a位点缺乏可调节的tRNA，这表明解码动力学受损。核糖体分析显示，低优化密码子富集于ccr4 -非结合细长核糖体的a位点。这一观察解释了低温电镜观察到的低a位点tRNA占用，并提出了与密码子最优性监测的联系。同样，通过mRNA稳定性分析，我们发现Not5的缺失导致mRNA降解机制无法感知密码子最优性。在Not5缺失、Not5 n端缺失、eS25缺失和Not4缺失eS7泛素化过程中检测到mRNA半衰期失调，这显然是Ccr4-Not在核糖体上进一步活性的上游先决条件。此外，在这些突变体中发现mRNA脱帽受损，这证实了在该途径中，Ccr4-Not触发最优性监测下游的脱帽。我们的分析阐明了介导mRNA衰变的Ccr4-Not复合体与核糖体之间的直接物理联系。当a位点由于解码动力学缓慢而缺乏tRNA时，Ccr4-Not复合体（通过Not5亚基）特异性地结合到核糖体e位点，这取决于Not4亚基先前对eS7的泛素化。核糖体的这种状态发生在a位点存在非最佳密码子的情况下，这解释了富含非最佳密码子的转录本的半衰期较短。因此，我们的研究结果通过Ccr4-Not复合体为翻译效率与mRNA稳定性的协调提供了机制见解。信使RNA （mRNA）衰减率的控制与翻译伸长密切相关，但这些事件的空间协调性尚不清楚。Ccr4-Not复合体通过死基化和脱帽激活启动mRNA衰变。我们使用冷冻电子显微镜、核糖体分析和mRNA稳定性分析相结合的方法来研究Ccr4-Not通过Not5亚基与酿酒酵母核糖体e位点的特异性相互作用募集到核糖体上。这种相互作用发生在核糖体缺乏可调节的a位点转移RNA时，表明低密码子最优性。相互作用的缺失导致mRNA降解机制无法感知密码子最优性。我们的研究结果阐明了Ccr4-Not复合物与核糖体之间的物理联系，并为解码效率与mRNA稳定性的耦合提供了机制见解。Ccr4-Not将翻译效率与mRNA降解结合起来。当核糖体遇到非最佳密码子时，低解码效率导致在同源tRNA被安置在a位点之前，e位点tRNA解离的可能性增加。因此，核糖体e位点采用一种特定的构象，通过其Not5亚基的n端被Ccr4-Not复合体识别，最终触发Xrn1降解mRNA。
The tightly controlled process of gene expression requires messenger RNAs (mRNAs), which represent DNA-derived blueprints for polypeptides, to be translated by the protein-producing machinery of the cell, the ribosomes. Therefore, protein levels depend largely on cellular mRNA levels, and the control of mRNA decay is one of the most critical processes for setting the overall level of gene expression. Half-lives of mRNAs vary greatly between different transcripts, and regulation of the mRNA decay rate is intimately connected to the elongation phase of mRNA translation. To that end, codon optimality has been established as a key parameter for determining mRNA half-life in multiple eukaryotic organisms. It has also been established that the timely decay of short-lived mRNAs enriched with nonoptimal codons requires the Ccr4-Not complex. Ccr4-Not is an essential protein complex, with its best understood role in mRNA degradation, where it serves as the major cytoplasmic 3′-poly(A)-tail deadenylase that initiates decay of most mRNAs. By deadenylation and subsequent activation of the mRNA decapping machinery, the Ccr4-Not complex renders mRNAs accessible to the major degrading exonucleases, such as Xrn1 on the 5′ end and the exosome on the 3′ end. The molecular mechanism underlying codon optimality monitoring and coordination with mRNA decay by the Ccr4-Not complex has remained elusive. Because nonoptimal codons affect decoding kinetics of the ribosome and mRNA degradation occurs largely cotranslationally, it is highly plausible that codon optimality is directly monitored on the ribosome. In addition, a direct physical link between the participating Ccr4-Not complex and the ribosome has been suggested previously, and the Not4 subunit of the complex, an E3 ligase, ubiquitinates the eS7 protein of the 40S ribosomal subunit in yeast. Therefore, we set out to gain insights into the connection between the Ccr4-Not complex and the translation machinery in the context of mRNA homeostasis by combining cryo–electron microscopy (cryo-EM), ribosome profiling, and biochemical analysis. We used affinity-purified native Ccr4-Not–ribosome complexes from Saccharomyces cerevisiae for analysis by cryo-EM and found that recruitment of Ccr4-Not to the ribosome occurs via the Not5 subunit. The N terminus of Not5—in particular, a three α-helix bundle—interacted specifically with the ribosomal E-site, and deletion of the Not5 N-terminus resulted in the loss of stable ribosome association of the Ccr4-Not complex. However, ubiquitination of the small ribosomal subunit protein eS7 through the Not4 subunit still occurred. The Not5 interaction involved the ribosomal protein eS25 of the small subunit, in addition to transfer RNA (tRNA) and ribosomal RNAs (rRNAs). We found that Ccr4-Not interacts with both initiating and elongating ribosomes. In either case, Not5 engaged the E-site only when the ribosome adopted a distinct conformation lacking accommodated tRNA in the A-site, indicative of impaired decoding kinetics. Ribosome profiling revealed that low-optimality codons were enriched in the A-site in the Ccr4-Not–bound elongating ribosomes. This observation explained the low A-site tRNA occupancy observed with cryo-EM and suggested a link to codon optimality monitoring. Consistently, using mRNA stability assays, we found that loss of Not5 resulted in the inability of the mRNA degradation machinery to sense codon optimality. The observed dysregulation of mRNA half-life was detected upon Not5 deletion, Not5 N-terminal deletion, eS25 deletion, and loss of eS7 ubiquitination by Not4, which apparently serves as an upstream prerequisite for further Ccr4-Not activity on the ribosome. In addition, mRNA decapping was found to be impaired in these mutants, which confirmed that, in this pathway, Ccr4-Not triggers decapping downstream of optimality monitoring. Our analysis elucidates a direct physical link between the mRNA decay–mediating Ccr4-Not complex and the ribosome. Dependent on preceding ubiquitination of eS7 by the Not4 subunit, the Ccr4-Not complex binds (via the Not5 subunit) specifically to the ribosomal E-site when the A-site lacks tRNA because of slow decoding kinetics. This state of the ribosome occurs in the presence of nonoptimal codons in the A-site, which explains the shorter half-lives of transcripts enriched in nonoptimal codons. Thus, our findings provide mechanistic insights into the coordination of translation efficiency with mRNA stability through the Ccr4-Not complex. Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo–electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in Saccharomyces cerevisiae. This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability. Ccr4-Not couples translation efficiency to mRNA degradation. When ribosomes encounter nonoptimal codons, low decoding efficiency leads to an increased likelihood of dissociation of the E-site tRNA before the cognate tRNA is accommodated in the A-site. As a result, the ribosomal E-site adopts a specific conformation, which is recognized by the Ccr4-Not complex through the N-terminus of its Not5 subunit, eventually triggering mRNA degradation by Xrn1.
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影响因子：
64.8
作者：
通讯作者：
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影响因子：
3.3
作者：
Parker, Roy
通讯作者：
Parker, Roy
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
16.6
作者：
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通讯作者：
Inada T