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。一些工作在2016年1月实验室启动后不久就开始了，并在实验室空间的完整设置和清洁完成后扩大，并雇用了单位成员。 研究集中在核糖体在终止密码子处完成翻译后如何分解以允许核糖体亚基重新用于新一轮翻译的问题上。这种亚基的循环是至关重要的，因为它的失败会导致核糖体的短缺，限制细胞制造蛋白质的能力。当终止密码子被典型的释放因子eRF1和eRF3解码时，该过程开始。在eRF3水解GTP后，ATP酶Rli1（高等真核生物中的ABCE1）将两个亚基彼此分离。在先前的工作中，我们确定细胞中缺乏足够的Rli 1导致核糖体在3 'UTR中的积累和短开放阅读框的翻译。已知再循环因子ABCE1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ABCE1的活性被认为是由基因调节的，这些基因被干扰素刺激上调。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。我们也对3'UTR核糖体的生物学作用感兴趣。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。早期的结果表明，我们可以刺激细胞的抗病毒反应，我们现在正在开发计算方法来分析这些细胞系的核糖体谱。最后，我们还开始设计结构，以利用新的单分子荧光技术对细胞中的单个mRNA进行成像翻译，例如SunTag和荧光标记的RNA茎环结合蛋白。

We broadly investigate the mechanisms used in cells to regulate gene expression at the translational level. Some work began soon after the lab's launch in January of 2016 and expanded once full setup and cleaning of the lab space was accomplished, and unit members were hired. Research has focused on the question of how ribosomes are disassembled following the completion of translation at stop codons to allow the ribosomal subunits to be reused for new rounds of translation. This recycling of subunits is critical because its failure can lead to a shortage of ribosomes, limiting the cell's ability to make protein. This process 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 sufficient Rli1 in the cell leads to an 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 stimulation. A better understanding of the mechanism of ribosome recycling is therefore important for overcoming major challenges to human health. 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. We are also interested in the biological role for 3'UTR ribosomes. 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. Early results have shown we can stimulate the cell's antiviral response and we now are developing computational methods for analyzing ribosome profiling of these cell lines. Finally, we have also begun designing constructs to take advantage of new single molecule fluorescence technologies for imaging translation on single mRNAs in cells, such as SunTag and fluorescently-labeled RNA stem loop binding proteins.