Molecular mechanism of the ribosome and functions of translational regulation

我们广泛地研究了细胞中用于在翻译水平上调节基因表达的机制。目前的研究主要集中在核糖体在终止密码子处完成翻译后如何分解的问题上。如果没有这个循环过程，核糖体将迅速积累在终止密码子上，限制细胞制造新蛋白质的能力。 该实验室主要采用高通量测序方法，如mRNA-Seq和核糖体足迹分析与计算分析。我们还使用了一系列的生化方法，如蛋白质印迹和报告分析，以补充这项工作。最后，我们正在开发的工具，活酵母和哺乳动物细胞中的单聚核糖体成像，使用荧光显微镜和suntag系统。 当终止密码子被典型的释放因子eRF 1和eRF 3解码时，再循环开始。在eRF 3水解GTP后，ATP酶Rli 1（高等真核生物中的ABCE 1）将两个亚基彼此分离。在先前的工作中，我们确定细胞中Rli 1的缺乏导致核糖体在3 'UTR中的惊人积累和短开放阅读框的翻译。已知再循环因子ABCE 1在许多类型的癌症中上调，这表明核糖体再循环在癌细胞中至关重要，可能是确保在快速增殖期间为新一轮翻译提供足够的再循环核糖体的一种方式。我们相信这个过程在先天免疫反应中也是至关重要的，因为ABCE 1的活性被认为是由干扰素上调的基因调节的。因此，更好地了解核糖体再循环的机制对于克服人类健康面临的重大挑战至关重要。 我们最近研究了酵母因子Tma 64、Tma 20和Tma 22（哺乳动物中的eIF 2D、MCT-1和DENR）。我们的工作现在强烈地表明，这些因子是核糖体循环所必需的。没有它们，我们发现核糖体进入3 'UTR并重新启动新的翻译，可能至少有两种机制。首先，我们发现了证据（3 'UTR AUG密码子上的核糖体谱峰和通过报告蛋白的蛋白质印迹），这些因子促进40 S核糖体的再循环。然而，我们也观察到80 S再起始和终止密码子通读的证据。因此，我们的研究表明，这些因素在多个水平上阻止3 'UTR的翻译中起着关键作用。这些因子的突变已被证明与人类的自闭症和癌症有关，这表明在酵母中缺乏这些因子的情况下产生的肽可能与这些疾病有关。 我们已经开始工作，破译的规则和因素参与核糖体重新启动下游的终止密码子，通过使用masspec和核糖体足迹分析方法。我们对报告基因构建体的研究表明，80 S核糖体能够在终止密码子附近的3 'UTR长区域上释放肽后容易地重新启动翻译。因此，回收机械的组件，如Rli 1，Tma 64，Tma 20和Tma 22，对于防止这种异常过程的发生至关重要。 我们还在探索3 'UTR核糖体的潜在生物学作用，以及在某些细胞环境下失去有效循环可以改变适应性的可能性。我们正在研究营养剥夺应激（酵母）和模拟病毒感染（人类细胞系）的影响，例如，在降低终止和回收效率方面。我们的结果现在表明，许多应激诱导3 'UTR的翻译，但所涉及的机制是可变的（40 S再起始，80 S再起始，通读等），这意味着被翻译的3 'UTR肽的库依赖于应激。我们还表明，我们可以刺激细胞的抗病毒反应，当这种情况发生时，3 'UTR核糖体的水平会增加。这一发现意味着在先天免疫应答期间激活的因子直接影响核糖体再循环，并且产生的肽可能具有免疫原性。

We broadly investigate the mechanisms used in cells to regulate gene expression at the translational level. Current research is focused on the question of how ribosomes are disassembled following the completion of translation at stop codons. Without this recycling process, ribosomes would quickly accumulate at stop codons, limiting the cell's ability to make new protein. The lab primarily employs high-throughput sequencing methods, such as mRNA-Seq and ribosome footprint profiling with computational analysis. We also use an array of biochemical approaches, such as western blots and reporter assays, to complement this work. Finally, we are developing tools for imaging single polysomes in living yeast and mammalian cells, using fluorescence microscopy and the suntag system. Recycling begins when the stop codon is decoded by the canonical release factors, eRF1 and eRF3. Following GTP hydrolysis by eRF3, the ATPase Rli1 (ABCE1 in higher eukaryotes) separates the two subunits from each other. In prior work, we established that lack of Rli1 in the cell leads to a surprising accumulation of ribosomes in the 3'UTR and the translation of short open reading frames. The recycling factor ABCE1 is known to be upregulated in many types of cancer, suggesting that ribosome recycling is critical in cancer cells, potentially as a way to ensure an adequate supply of recycled ribosomes for new rounds of translation during rapid proliferation. We believe this process to also be critical during the innate immune response because the activity of ABCE1 is thought to modulated by genes that are upregulated by interferon. A better understanding of the mechanism of ribosome recycling is therefore important for overcoming major challenges to human health. We have recently investigated the yeast factors Tma64, Tma20, and Tma22 (eIF2D, MCT-1, and DENR in mammals). Our work has now strongly suggested that these factors are required for ribosome recycling. Without them, we have found that ribosomes enter 3'UTRs and reinitiate new translation, likely by at least two mechanisms. First, we found evidence (ribosome profiling peaks on 3'UTR AUG codons and via western blot of reporter proteins) that that these factors promote recycling of 40S ribosomes. However, we also observed evidence of 80S reinitiation and stop codon readthrough. Our research has therefore shown that these factors play a critical role in preventing translation of 3'UTRs at multiple levels. Mutation of these factors has been shown to be associated with autism and cancer in humans, suggesting that the peptides produced in their absence in yeast may be linked to these diseases. We have begun work on deciphering the rules and factors involved in ribosome reinitiation downstream of the stop codon by using masspec and ribosome footprint profiling approaches. Our research on reporter constructs suggests that 80S ribosomes are readily capable of reinitiating translation following peptide release over a long region of the 3'UTR adjacent to the stop codon. Therefore, components of the recycling machinery such as Rli1, Tma64, Tma20, and Tma22 are critical to prevent this aberrant process from occurring. We are also exploring potential biological roles for 3'UTR ribosomes and the possibility that loss of efficient recycling under some cellular environments can alter fitness. We are examining the effects of nutrient deprivation stress (yeast) and simulated viral infection (human cell lines), for example, in reducing the efficiency of termination and recycling. Our results now suggest many stresses induce translation of 3'UTRs but that the mechanism involved is variable (40S reinitiation, 80S reinitiation, readthrough, etc.), implying that the repertoire of 3'UTR peptides that is translated depends on the stress. We have also shown that we can stimulate the cell's antiviral response and that levels of 3'UTR ribosomes increase when this occurs. This finding implies that factors activated during the innate immune response directly affect ribosome recycling and that peptides produced may be immunogenic.