Understanding genome-wide and single-molecule dynamics of colliding ribosomes during health and disease

了解健康和疾病期间碰撞核糖体的全基因组和单分子动力学

• 批准号: 10026314
• 负责人: Fatma Sezen Meydan Marks
• 金额: --
• 依托单位: U.S. NATIONAL INST DIABETES/DIGST/KIDNEY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10026314
• 关键词: Cells; Cellular Stress; Color; Complex; Defect; Disease; Engineering; Event; FMR1; FMRP; Fragile X Syndrome; Goals; Health; Homeostasis; Human; Human Pathology; Impairment; Inherited; Intellectual functioning disability; Kinetics; Link; Messenger RNA; Methods; Modeling; Monitor; Nature; Neurodevelopmental Disorder; Neuronal Differentiation; Neurons; Organism; Pathologic; Pathway interactions; Patients; Phenotype; Physiological; Physiology; Prevalence; Process; Protein Biosynthesis; Proteins; Quality Control; Regulation; Ribosomes; Role; Series; Study models; System; Techniques; Testing; Therapeutic; Time; Transcript; Translating; Translations; Yeasts; biological adaptation to stress; disease phenotype; experimental study; genome-wide; inhibitor/antagonist; mRNA Decay; microscopic imaging; molecular imaging; neurodevelopment; novel therapeutics; prevent; protein expression; real time monitoring; single molecule; transcriptome

PROJECT SUMMARY/ABSTRACT Eukaryotic ribosomes translating defective mRNAs, such as those that are damaged, “stall” and become unavailable for new rounds of translation. The ribosome-associated quality control (RQC) system detects the stalled complex formed by two collided ribosomes (“disome”) and degrades the defective mRNAs to prevent aberrant translation. Since this pathway leads to irreversible mRNA decay, it is critical for the RQC machinery to differentiate functional ribosome pausing events, from aberrant ribosome stalling cases that need to be resolved. However, previous RQC studies used artificial mRNA substrates that induce extreme cases of ribosome stalling. Therefore, it is unknown how frequently and where disomes form on regular transcripts. It is also unclear if RQC recognizes all disomes and what the cellular consequences of recognition are. Interestingly, impaired ribosome collisions have been linked to a neurodevelopmental disorder, Fragile-X syndrome (FXS), which is the most common form of inherited intellectual disability. Nevertheless, the pathological dynamics of ribosome collisions during FXS as well as the interplay between collisions and RQC pathway during neurodevelopment have been poorly studied. There is a critical need, therefore, to determine the dynamics of ribosome collisions in healthy cells and to understand how their dysregulation leads to FXS. The objective of this proposal is to determine the role of ribosome collisions during neurodevelopment. My hypothesis is that under physiological conditions, RQC-targeted disomes form on regular transcripts and they maintain neuronal homeostasis by regulating protein expression. I further postulate that dysregulation of disome formation leads to cellular stress and causes disease phenotypes. To test this idea, in aim 1, I will determine the genome-wide distribution of disomes in human cells using the Disome-seq technique that we recently established in our lab. To visualize the dynamics of RQC upon ribosome collision, in aim 2, I will monitor real-time regulation of RQC using cutting-edge dual-color single molecule imaging in human cells. To understand the functional role of ribosome collisions, in aim 3, I will characterize the link between dysregulated ribosome stalling in an FXS model of neuronal differentiation. I will further study the action of translation inhibitors for their potential of restoring the collisions in neurons. Overall, the proposed studies aim to determine the prevalence of ribosome collisions in the cell and how dysregulation of these collisions can cause neurodevelopmental defects.

项目摘要/摘要 真核核糖体翻译有缺陷的mRNAs，例如那些受损的mRNA， “拖延”，无法进行新一轮的翻译。核糖体相关质量 控制(RQC)系统检测由两个相互碰撞的核糖体(“二体”)形成的停滞的复合体 并降解有缺陷的mRNAs以防止异常翻译。因为这条路通向 不可逆的mRNA衰退，对RQC机制来说，分化功能核糖体是至关重要的 暂停事件，来自需要解决的异常核糖体停滞病例。然而， 以前的RQC研究使用的是人工mRNA底物，可以诱导极端情况下的核糖体 在拖延时间。因此，在常规转录本上形成二倍体的频率和位置尚不清楚。 目前还不清楚RQC是否识别所有的胞体，以及识别的细胞后果是什么 是。有趣的是，受损的核糖体碰撞与神经发育有关 精神障碍，脆性X综合征(FXS)，这是最常见的遗传性智力障碍 残疾。然而，FXS过程中核糖体碰撞的病理动力学以及 在神经发育过程中，碰撞和RQC通路之间的相互作用一直很差。 学习。因此，迫切需要确定核糖体碰撞的动力学。 健康的细胞并了解它们的失调是如何导致FXS的。 这项建议的目标是确定核糖体碰撞在 神经发育。我的假设是，在生理条件下，RQC靶向的脱氧核糖核酸 在正常转录本上形成，它们通过调节蛋白质来维持神经元的动态平衡 表情。我进一步假设，二体形成的失调会导致细胞应激和 会引起疾病表型。为了测试这个想法，在目标1中，我将确定全基因组 用我们新近研究的二体序列技术研究二体在人类细胞中的分布 在我们的实验室建立。为了可视化核糖体碰撞时RQC的动力学，在目标2中，我将 用尖端双色单分子成像监测RQC的实时调节 人类细胞。为了理解核糖体碰撞的功能作用，在目标3中，我将描述 在神经元分化的FXS模型中，失调核糖体停滞之间的联系。这就做 进一步研究翻译抑制物在恢复碰撞中的作用 神经元。总体而言，拟议的研究旨在确定核糖体碰撞在 以及这些碰撞的失调如何导致神经发育缺陷。

项目成果

A cellular handbook for collided ribosomes: surveillance pathways and collision types.

• DOI: 10.1007/s00294-020-01111-w
• 发表时间: 2021-03
• 期刊: Current genetics
• 影响因子: 2.5
• 作者: Meydan S;Guydosh NR
• 通讯作者: Guydosh NR

40S ribosome profiling reveals distinct roles for Tma20/Tma22 (MCT-1/DENR) and Tma64 (eIF2D) in 40S subunit recycling.

40S 核糖体分析揭示了 Tma20/Tma22 (MCT-1/DENR) 和 Tma64 (eIF2D) 在 40S 亚基回收中的不同作用。

• DOI: 10.1038/s41467-021-23223-8
• 发表时间: 2021-05-20
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Young DJ;Meydan S;Guydosh NR
• 通讯作者: Guydosh NR

Is there a localized role for translational quality control?

翻译质量控制是否有本地化的角色？

• DOI: 10.1261/rna.079683.123
• 发表时间: 2023-11
• 期刊: RNA (New York, N.Y.)

mRNA Location and Translation Rate Determine Protein Targeting to Dual Destinations.

mRNA 位置和翻译率决定蛋白质靶向双重目的地。

• DOI: 10.1101/2023.04.24.538105
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Gasparski,AlexanderN;Moissoglu,Konstadinos;Pallikkuth,Sandeep;Meydan,Sezen;Guydosh,NicholasR;Mili,Stavroula
• 通讯作者: Mili,Stavroula

Context-specific action of macrolide antibiotics on the eukaryotic ribosome.

• DOI: 10.1038/s41467-021-23068-1
• 发表时间: 2021-05-14
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Svetlov MS;Koller TO;Meydan S;Shankar V;Klepacki D;Polacek N;Guydosh NR;Vázquez-Laslop N;Wilson DN;Mankin AS
• 通讯作者: Mankin AS