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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/文摘