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:
- 金额:$ 6.95万
- 依托单位:
- 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:
- 关键词:AntibodiesAuxinsBiogenesisBiological AssayBirthBreast Epithelial CellsCell NucleolusCell NucleusCell physiologyCellsChildClinicalCrystalline LensDataDefectDiagnosisDiseaseDown-RegulationDysmorphologyEukaryotaEukaryotic CellFunctional disorderGenetic DiseasesGenetic TranscriptionHistonesHumanHuman GeneticsIntellectual functioning disabilityKnowledgeLaboratoriesLifeMCF10A cellsMalignant NeoplasmsMeasuresMediatingMembraneMessenger RNAMetabolismMethodologyMethodsMethylationMethyltransferaseMicrocephalyMitochondrial ProteinsMorphologyNatureNeurodevelopmental ImpairmentOrganellesPathogenesisPatientsPhenotypeProcessProtein Synthesis InhibitionProteinsProteomicsRNA ProcessingRNA methylationRNA, Ribosomal, 18SRegulationResearchResistanceRibosomal DNARibosomal ProteinsRibosomal RNARibosomesRoleS-AdenosylhomocysteineS-AdenosylmethionineSeizuresSeriesSmall Interfering RNASulfateSulfitesSurfaceSystemTestingTherapeuticTranslatingUridineValidationVariantWestern BlottingWorkclinically relevantcraniofacialdevelopmental diseaseexperimental studyfibrillaringenome wide screengenome-widehistone methylationhuman diseaseinnovationloss of functionmetabolomicsnext generationnovelribosomopathysulfite 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。
项目成果
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