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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.

项目总结/摘要线粒体蛋白亚硫酸盐氧化酶（SUOX）已令人惊讶地成为核糖体的潜在调节剂在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节过程。尽管它的重要性和相关性的疾病，与癌症和核糖体病一样，人细胞中核糖体生物发生的调节还没有完全了解。为了确定这一过程的新调节因子，Baserga实验室率先进行了全基因组siRNA筛选使用每个细胞核的核仁数作为终点。MCF 10A乳腺上皮细胞平均核仁数为2-3，减少到1表明异常的核糖体生物发生。SUOX是一个一种线粒体蛋白质，它意外地在这个屏幕上出现。SUOX氧化毒性细胞已知某些SUOX变异体会导致严重的、致命的发育性疾病孤立性亚硫酸氧化酶缺乏症（ISOD）。验证表明，SUOX耗竭减少核仁数和核糖体RNA（rRNA）生物发生。我收集了更多的数据来证明核糖体的生物合成蛋白质组学数据强烈支持SUOX在制造核糖体ISOD与已知的核糖体病呈现的相似性，沿着我们的有趣的初步结果，提出了令人信服的问题，核糖体生物合成参与 ISOD发病机制。深入到机制层面，我们的初步代谢组学数据显示， SUOX耗竭导致核仁甲基转移酶原纤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，分别到目前为止，还没有研究SUOX在核糖体生物合成中的作用，也没有研究核糖体的 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确切作用，利用已建立的测定方法测量该过程的多个阶段。我将描述使用最近开发的RibOxi-seq方法对rRNA甲基化的SUOX消耗，以及组蛋白使用已建立的抗体进行甲基化。我将进一步验证我们的结果使用生长素诱导降解决定子版本2（AID 2）系统。在目标2中，我将确定疾病相关的SUOX变体对人类的影响。通过用siRNA修复介导的SUOX耗尽后发生的缺陷来实现核糖体生物合成功能性沉默和疾病相关的功能丧失SUOX变体。提出的实验将明确定义SUOX在人类核糖体生物合成中的作用，并描述已知疾病的影响- 导致这一基本细胞过程的变异。我们独特的方法基于对核仁功能，结合成熟的和新的方法来研究核糖体生物发生，使我们有机会将SUOX研究领域带入一个全新的方向，了解SUOX在细胞代谢和人类遗传疾病中的功能。