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Defining a role for the mitochondrial protein sulfite oxidase in nucleolar ribosome biogenesis

定义线粒体蛋白亚硫酸氧化酶在核仁核糖体生物合成中的作用

基本信息

批准号：
10750182
负责人：
Emily Catherine Sutton
金额：
$ 6.95万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10750182
关键词：
Antibodies Auxins Biogenesis Biological Assay Birth Breast Epithelial Cells Cell Nucleolus Cell Nucleus Cell physiology Cells Child Clinical Crystalline Lens Data Defect Diagnosis Disease Down-Regulation Dysmorphology Eukaryota Eukaryotic Cell Functional disorder Genetic Diseases Genetic Transcription Histones Human Human Genetics Intellectual functioning disability Knowledge Laboratories Life MCF10A cells Malignant Neoplasms Measures Mediating Membrane Messenger RNA Metabolism Methodology Methods Methylation Methyltransferase Microcephaly Mitochondrial Proteins Morphology Nature Neurodevelopmental Impairment Organelles Pathogenesis Patients Phenotype Process Protein Synthesis Inhibition Proteins Proteomics RNA Processing RNA methylation RNA, Ribosomal, 18S Regulation Research Resistance Ribosomal DNA Ribosomal Proteins Ribosomal RNA Ribosomes Role S-Adenosylhomocysteine S-Adenosylmethionine Seizures Series Small Interfering RNA Sulfate Sulfites Surface System Testing Therapeutic Translating Uridine Validation Variant Western Blotting Work clinically relevant craniofacial developmental disease experimental study fibrillarin genome wide screen genome-wide histone methylation human disease innovation loss of function metabolomics next generation novel ribosomopathy sulfite oxidase

项目摘要

PROJECT SUMMARY/ABSTRACT The mitochondrial protein sulfite oxidase (SUOX) has surprisingly emerged as a potential regulator of ribosome biogenesis in a genome-wide screen. Ribosome biogenesis, which occurs in the nucleolus of eukaryotic cells, is a highly regulated process essential for cell function. Despite its importance and relevance to diseases such as cancer and ribosomopathies, the regulation of ribosome biogenesis in human cells is not fully understood. To identify novel regulators of this process, the Baserga laboratory pioneered a genome-wide siRNA screen using the number of nucleoli per nucleus as an endpoint. MCF10A breast epithelial cells have an average nucleolar number of 2-3, and a decrease to one indicates aberrant ribosome biogenesis. SUOX is a mitochondrial protein that has unexpectedly surfaced as a hit from this screen. SUOX oxidizes toxic cellular sulfite to sulfate, and some SUOX variants are known to cause the severe, fatal developmental disease Isolated Sulfite Oxidase Deficiency (ISOD). Validation shows that SUOX depletion reduces nucleolar number and ribosomal RNA (rRNA) biogenesis. I have collected further data demonstrating ribosome biogenesis disruption upon SUOX depletion, and proteomics data strongly supporting a role for SUOX in making ribosomes. The similarity of the presentation of ISOD to that of known ribosomopathies, along with our intriguing preliminary results, has raised compelling questions about the involvement of ribosome biogenesis in ISOD pathogenesis. Drilling down to the mechanistic level, our preliminary metabolomics data reveal that SUOX depletion causes a decrease in the methyl donor required for the nucleolar methyltransferase fibrillarin. Fibrillarin methylates an rDNA-specific histone and rRNA, regulating rDNA transcription and rRNA processing, respectively. To date, no work has investigated the role of SUOX in ribosome biogenesis nor of ribosome biogenesis in ISOD pathogenesis. In Aim 1, I will establish the precise role of SUOX in ribosome biogenesis, taking advantage of established assays measuring multiple stages of the process. I will describe effects of SUOX depletion on rRNA methylation using the recently developed RibOxi-seq method, and histone methylation using established antibodies. I will further validate our results using the auxin-inducible degron version 2 (AID2) system. In Aim 2, I will determine the effects of disease-associated SUOX variants on human ribosome biogenesis by rescuing the defects that occur upon siRNA-mediated SUOX depletion with translationally silent and disease-associated loss-of-function SUOX variants. The experiments proposed will clearly define a role for SUOX in human ribosome biogenesis and describe the effects of known disease- causing variants on this essential cellular process. Our unique approach based on an unbiased screen for nucleolar function, combined with both well-established and novel methodology to study ribosome biogenesis, gives us the opportunity to take the field of SUOX research in an entirely new direction that will open avenues for understanding the function of SUOX in cellular metabolism and human genetic disease.

项目概要/摘要线粒体蛋白亚硫酸氧化酶 (SUOX) 令人惊讶地成为核糖体的潜在调节剂全基因组筛选中的生物发生。核糖体生物发生发生在真核细胞的核仁中，是细胞功能所必需的高度调控的过程。尽管它对疾病具有重要意义和相关性，与癌症和核糖体病一样，人类细胞中核糖体生物发生的调节尚不完全清楚。为了识别这一过程的新调节因子，Baserga 实验室率先进行了全基因组 siRNA 筛选使用每个核的核仁数量作为终点。 MCF10A 乳腺上皮细胞的平均核仁数量为 2-3，减少至 1 表明核糖体生物发生异常。 SUOX是一个线粒体蛋白出人意料地在这个屏幕上大受欢迎。 SUOX 氧化有毒细胞亚硫酸盐转化为硫酸盐，一些 SUOX 变体已知会导致严重、致命的发育疾病孤立性亚硫酸氧化酶缺乏症（ISOD）。验证表明 SUOX 耗尽会减少核仁数量和核糖体 RNA (rRNA) 生物发生。我收集了更多证明核糖体生物发生的数据 SUOX 耗尽后的破坏，蛋白质组学数据强烈支持 SUOX 在制造中的作用核糖体。 ISOD 的表现与已知核糖体病的相似性，以及我们的研究有趣的初步结果，提出了关于核糖体生物发生参与的令人信服的问题 ISOD发病机制。深入到机制层面，我们的初步代谢组学数据表明 SUOX 耗尽会导致核仁甲基转移酶纤维蛋白所需的甲基供体减少。 Fibrillarin 甲基化 rDNA 特异性组蛋白和 rRNA，调节 rDNA 转录和 rRNA 加工，分别。迄今为止，还没有研究研究 SUOX 在核糖体生物合成中的作用，也没有研究过核糖体的作用 ISOD 发病机制中的生物发生。在目标 1 中，我将确定 SUOX 在核糖体生物发生中的精确作用，利用测量过程多个阶段的既定测定法。我将描述效果使用最近开发的 RibOxi-seq 方法和组蛋白消除 rRNA 甲基化的 SUOX 使用已建立的抗体进行甲基化。我将使用生长素诱导的降解决定子进一步验证我们的结果版本 2 (AID2) 系统。在目标 2 中，我将确定与疾病相关的 SUOX 变异对人类的影响通过挽救 siRNA 介导的 SUOX 耗尽时发生的缺陷来实现核糖体生物合成翻译沉默和疾病相关的功能丧失 SUOX 变异。所提出的实验将明确定义了 SUOX 在人类核糖体生物合成中的作用，并描述了已知疾病的影响- 导致这一重要的细胞过程发生变异。我们基于公正筛选的独特方法核仁功能，结合成熟和新颖的方法来研究核糖体生物发生，让我们有机会将 SUOX 研究领域推向一个全新的方向，这将开辟新的道路了解 SUOX 在细胞代谢和人类遗传疾病中的功能。