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

• 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
• 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

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.

项目摘要/摘要 线粒体蛋白亚硫酸盐氧化酶(SUOX)令人惊讶地成为核糖体的潜在调节因子。 全基因组屏幕上的生物发生。核糖体生物发生，发生在真核细胞的核仁中， 是一个高度受调控的过程，对细胞功能至关重要。尽管它的重要性和相关性与诸如 作为癌症和核糖病，核糖体生物发生的调节在人类细胞中还不完全清楚。 为了确定这一过程的新调控因素，巴塞加实验室率先进行了全基因组siRNA筛查 以每个核的核仁数量为终点。MCF10A乳腺上皮细胞平均有 核仁数目为2-3，核仁数目减少到1表示核糖体生物发生异常。SUOX是一种 线粒体蛋白质出人意料地从这个屏幕上走红。SUOX氧化有毒细胞 亚硫酸盐到硫酸盐，以及一些SUOX变体已知会导致这种严重的、致命的发育性疾病 孤立性亚硫酸盐氧化酶缺乏症(Isod)。验证表明，SUOX耗竭减少了核仁数量 和核糖体RNA(RRNA)的生物发生。我收集了更多的数据来证明核糖体的生物发生 对SUOX耗竭的破坏，以及蛋白质组学数据有力地支持SUOX在 核糖体。Isod的表现与已知核糖病的相似之处，以及我们的 耐人寻味的初步结果，提出了关于核糖体生物发生参与 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耗尽会导致核仁甲基转移酶原纤维蛋白所需的甲基供体减少。 纤维素甲基化rDNA特异组蛋白和rRNA，调节rDNA转录和rRNA加工， 分别进行了分析。到目前为止，还没有研究SUOX在核糖体生物发生中的作用或核糖体的作用 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确切作用， 利用已建立的检测方法测量该过程的多个阶段。我将描述一下 用最近开发的RibOxi-Seq方法和组蛋白来耗尽对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可降解蛋白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变体对人类的影响 修复siRNA介导的SUOX耗竭所产生的缺陷的核糖体生物发生 翻译沉默和疾病相关的功能丧失的SUOX变体。拟议中的实验将 明确定义SUOX在人类核糖体生物发生中的作用，并描述已知疾病的影响- 导致了这一重要细胞过程的变异。我们独特的方法基于无偏见的屏幕 核仁功能，结合成熟的和新的方法来研究核糖体的生物发生， 使我们有机会将SUOX研究领域带到一个全新的方向，这将开辟 以了解SUOX在细胞代谢和人类遗传病中的功能。