Molecular mechanism of the ribosome and functions of translational regulation

为了研究如何调节翻译以控制基因表达，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。我们也正在开发工具，通过使用荧光显微镜和由阵列GFP分子（SunTag）组成的报告分子，在活酵母和哺乳动物细胞中成像单聚核糖体。我们还通过对40 S（而不是80 S）核糖体或两个80 S核糖体（二体）之间的碰撞进行足迹分析来实现专门形式的核糖体分析。我们已经表明，40 S足迹通常发现在起始密码子，其中40 S亚基等待与60 S亚基连接，并在终止密码子，翻译完成后，60 S亚基已被删除。我们现在可以用酵母中的突变来增强这些信号，这些突变减缓了起始密码子处的60 S连接或终止密码子处的40 S亚基的解离。然后，我们使用这种方法来检测非规范开放阅读框架上的神秘翻译。相比之下，二体足迹法检测两个80 S核糖体在翻译过程中碰撞的情况，导致足迹是单个核糖体保护的足迹的两倍长。我们的工作揭示了二体形成是常见的，并且在随机和程序化的核糖体停滞事件中发生。有趣的是，我们还发现，当我们抑制翻译完成后去除核糖体的再循环过程时，二聚体在终止密码子处形成，并可以移动到3 'UTR。这种移动的二体可能对清除停滞的核糖体更广泛地至关重要。 核糖体（二体）之间的碰撞通常被认为是应激的信号，并且已知会招募核糖体质量控制（RQC）因子，如E3泛素连接酶Hel 2/ZNF 598（酵母/哺乳动物），其促进其分解，触发mRNA降解，并更广泛地激活应激反应途径。这一途径的丧失导致有毒肽的产生和神经变性。然而，核糖体在翻译过程中会自然地发生碰撞，这就提出了一个问题，即细胞是否能够区分有害的二聚体和执行功能的二聚体。为了解决这个问题，我们使用二体分析在全球范围内确定了核糖体碰撞的位点。有趣的是，我们发现所有的二体都被RQC途径识别，并表明Hel 2的丢失减少了二体的形成。因此，该途径被广泛用于检测许多类别的二体。我们的工作还表明，Hel 2不是碰撞核糖体的唯一传感器。我们发现，eIF 2 α激酶Gcn 2被激活时，Hel 2丢失。此外，我们发现，传统的触发Gcn 2，核糖体失速诱导的氨基酸饥饿，也检测到Hel 2。因此，我们已经确定，核糖体碰撞是通过多种应激传感途径检测到的，这些信号的整合可能有助于细胞对有害的核糖体碰撞和功能性核糖体碰撞做出不同的反应。 就像二体形成事件触发RQC途径一样，过早的翻译终止事件也触发称为无义介导的衰变（NMD）的应激途径，其导致mRNA的降解。该途径由编码突变的异常转录物触发，所述突变编码序列内的提前终止密码子。然而，已知NMD途径靶向许多明显正常的转录物，因此被认为在基因调控中发挥额外的作用。为了寻找在正常细胞中会导致过早翻译终止的隐蔽翻译事件，我们采用了40 S核糖体分析、RNA-Seq和其他方法。我们在编码序列内部发现了这样的事件，表明泄漏扫描，其中40 S核糖体未能找到主要的AUG起始密码子，而是启动下游翻译。我们还发现了长未解码转录异构体（LUTI）翻译的证据。在这些情况下，在正常条件下使用远上游启动子来产生编码上游开放阅读框（uORF）的长转录物。这些uORF翻译后的过早终止导致NMD，表明NMD对于完全沉默这些转录物是重要的。我们现在正在探索环境条件的变化如何改变启动子的使用，以支持uORF不转录的异构体，允许主要ORF被翻译并防止NMD。 我们的工作还研究了翻译是如何受到RNase L（先天免疫反应的一个分支）激活的影响。已知RNA酶L激活通过转录物的核酸内切裂解引起广泛的mRNA衰变。转录组如何翻译尚不清楚。我们在RNase L激活的细胞上使用核糖体分析，发现3 'UTR区域的核糖体占有率强烈增加。虽然人们认为RNase L可以调节翻译终止和再循环过程，因此产生这些效应，但我们的数据与这一假设不一致。相反，我们观察到非编码区的翻译增加，包括编码序列中的5 'UTR和框外ORF。此外，该作用还取决于RNase L的切割活性。解释这些结果的一个模型是核糖体可以启动mRNA衰变片段的翻译并翻译编码的ORF。我们计划进一步研究从这个过程中产生的肽如何与先天免疫反应相关，以及这些翻译事件是否影响这些片段的稳定性或拟议功能，例如细胞中dsRNA传感器的激活。 最后，我们还与Yihong Ye的实验室合作，揭示了ufmylation是一种重要的核糖体修饰，用于ER特异性RQC通路。

To investigate how translation is regulated to control gene expression, the lab has primarily employed high-throughput sequencing methods, such as mRNA-Seq and ribosome footprint profiling with computational analysis. We are also developing tools for imaging single polysomes in living yeast and mammalian cells by using fluorescence microscopy and reporters consisting of arrayed GFP molecules (SunTag). We also have implemented specialized forms of ribosome profiling by footprinting the 40S, as opposed to 80S, ribosome or collisions between two 80S ribosomes (disomes). We have shown that 40S footprints are typically found at start codons, where the 40S subunit waits to join with the 60S subunit, and at stop codons, after translation is complete and the 60S subunit has been removed. We can now enhance these signals with mutations in yeast that slow down 60S joining at start codons or dissociation of 40S subunits at stop codons. We then use this approach this to detect cryptic translation on non-canonical open reading frames. In contrast, disome footprinting detects cases where two 80S ribosomes collide during translation, leading to a footprint that is twice as long as that protected by a single ribosome. Our work has revealed that disome formation is common and occurs both stochastically and at programmed ribosome stalling events. Intriguingly, we also found that disomes form at stop codons and can move into the 3'UTR when we inhibit the recycling process that removes ribosomes after translation is complete. Such moving disomes may be more broadly critical for clearing stalled ribosomes. Collisions between ribosomes (disomes) are generally thought to be a signal of stress and are known to recruit ribosome quality control (RQC) factors, such as the E3 ubiquitin ligase Hel2/ZNF598 (yeast/mammals), that promote their resolution, trigger mRNA decay, and more broadly activate stress response pathways. Loss of this pathway leads to the production of toxic peptides and neurodegeneration. However, ribosomes naturally collide during the course of translation, raising the question of whether the cell can distinguish disomes that are harmful from those that perform a function. To address this, we globally identified sites where ribosomes collide using disome profiling. Intriguingly, we found that all disomes are recognized by the RQC pathway and showed that loss of Hel2 reduces disome formation. This pathway therefore is widely used to detect many classes of disomes. Our work also revealed that Hel2 is not the only sensor of collided ribosomes. We found that the eIF2alpha kinase Gcn2 is activated when Hel2 is lost. Moreover, we found that the traditional trigger of Gcn2, ribosome stalling induced by starvation of amino acids, was also detected by Hel2. We therefore have established that ribosome collisions are detected by multiple stress-sensing pathways and the integration of these signals may help the cell to differentially respond to harmful vs functional ribosome collisions. Much as disome formation events trigger the RQC pathway, premature translation termination events also trigger a stress pathway called nonsense-mediated decay (NMD) that leads to degradation of the mRNA. This pathway is triggered by aberrant transcripts that encode mutations that encode a premature stop codon within a coding sequence. However, the NMD pathway is known to target many apparently normal transcripts and is therefore thought to play additional roles in gene regulation. To search for cryptic translation events that would cause premature translation termination in normal cells, we employed 40S ribosome profiling, RNA-Seq, and other approaches. We found such events internal to coding sequences, indicative of leaky scanning, where the 40S ribosome fails to find the main AUG start codon and instead initiates translation downstream. We also found evidence for translation of long undecoded transcript isoforms (LUTIs). In these cases, far upstream promoters are used under normal conditions to make long transcripts that encode upstream open reading frames (uORFs). Premature termination after translation of these uORFs results in NMD, suggesting that NMD is important for fully silencing these transcripts. We are now exploring how changes in environmental conditions change promoter usage to favor of isoforms where the uORFs are not transcribed, allowing the main ORF be translated and preventing NMD. Our work has also examined how translation is influenced by activation of RNase L, a branch of the innate immune response. RNase L activation is known to cause widespread mRNA decay via endonucleolytic cleavage of transcripts. How the resultant transcriptome is translated is unknown. We used ribosome profiling on RNase L activated cells and found a strong increase in ribosome occupancy in 3'UTR regions. While it was thought that RNase L can modulate the translation termination and recycling processes and therefore give rise to these effects, our data are inconsistent with this hypothesis. Instead, we observe increased translation in non-coding regions generally, including 5'UTRs and out-of-frame ORFs in coding sequences. Moreover, the effects are dependent on the cleavage activity of RNase L. One model to explain these results is that ribosomes can initiate translation on mRNA decay fragments and translate the encoded ORFs. We plan to further investigate how peptides produced from this process are relevant to the innate immune response and whether these translation events affect the stability or proposed functions of these fragments, such as activation of dsRNA sensors in the cell. Finally, we also collaborated with lab of Yihong Ye to reveal that ufmylation is an important ribosome modification that is used for a ER-specific RQC pathway.