Regulation and Function of snoRNA Genes

snoRNA基因的调控和功能

• 批准号: 8755989
• 负责人: Wendy Victoria Gilbert
• 金额: $ 26.74万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8755989
• 关键词: Affect; Amino Acids; Biological Process; Biology; Breast; Cancer Patient; Cells; Cellular Stress; Codon Nucleotides; Colorectal Cancer; Data; Enzymes; Eukaryota; Functional RNA; Functional disorder; Future; Genes; Genetic; Genetic Code; Guide RNA; Health; Heat-Shock Response; Human; Lead; Length; Malignant Neoplasms; Malignant neoplasm of brain; Malignant neoplasm of lung; Malignant neoplasm of prostate; Maps; Mass Spectrum Analysis; Messenger RNA; Methods; Methylation; Modeling; Modification; Molecular Target; Multiple Myeloma; Nuclear; Nucleotides; Nutrient; Organism; Oxidative Stress; Pattern; Positioning Attribute; Pseudouridine; Reading; Regulation; Ribosomal RNA; Ribosomes; Risk; Sense Codon; Site; Small Nucleolar RNA; Specificity; Techniques; Terminator Codon; Testing; Therapeutic; Time; Translating; Translations; Treatment-Related Cancer; United States; Validation; Woman; Work; Yeasts; deprivation; design; environmental change; genome-wide; killings; leukemia; men; mutant; public health relevance; research study; response

DESCRIPTION (provided by applicant): snoRNAs are highly conserved non-coding RNAs that direct two types of site-specific post-transcriptional modifications of other RNAs - pseudouridinylation and 2'-O-methylation. Dysregulation of specific snoRNAs is associated with increased risk for breast, prostate, lung, brain and colorectal cancers as well as leukemia and multiple myeloma, but the mechanistic connections between altered snoRNA function and cancer are not understood. Recent discoveries herald a paradigm shift in the snoRNA field. First, snoRNAs were shown to have a greatly expanded list of potential RNA targets, including mRNAs. Second, some snoRNA-directed modifications were shown to be regulated by environmental changes. Finally, the presence of pseudouridine was shown to change how the genetic code is interpreted by ribosomes. Together, these results suggest potentially widespread regulatory functions for snoRNAs through directing post-transcriptional modification of mRNAs. We hypothesize that snoRNAs direct physiologically relevant pseudouridinylation of mRNA targets by a mechanism that involves relaxed specificity pairing between guide sequence and target RNA. To test this hypothesis, we have developed an efficient method for mapping the sites of snoRNA-directed pseudouridinylation genome-wide with single nucleotide precision (Pseudo-seq). Our preliminary results from Pseudo-seq experiments provide the first evidence that endogenous mRNAs are pseudouridinylated. Thus, we are uniquely well positioned to investigate this new paradigm for snoRNA function. In Aim 1 we will identify the regulated targets of snoRNA-directed pseudouridinylation in response to nutrient deprivation, oxidative stress and heat shock using Pseudo-seq. Mutants in the catalytic component of snoRNA enzymes, Cbf5/Dyskerin, will be used to determine which pseudouridines are formed by a snoRNA-dependent mechanism. Completion of this aim will comprehensively identify mRNA targets of regulated snoRNA-dependent modification in yeast. Aim 2 will use computational and targeted genetic approaches to dissect the sequence requirements for regulation by snoRNAs. Completion of this aim will facilitate accurate prediction of snoRNA targets in all eukaryotic organisms. Finally, Aim 3 will use mass spectroscopy to determine the functional consequences of mRNA pseudouridinylation for decoding by the ribosome. Completion of this aim has the potential to reveal substantial rewiring of the genetic code. Human health relevance: The results of this study will reveal new biological functions of snoRNAs and thereby illuminate the potential mechanisms by which snoRNA dysfunction leads to cancer. We believe that in the future, a better understanding of the function of snoRNAs in cells may lead to better treatment of cancers associated with perturbation of the function of snoRNA genes.

描述（由申请人提供）：snoRNA 是高度保守的非编码 RNA，可指导其他 RNA 的两种类型的位点特异性转录后修饰 - 假尿苷酰化和 2'-O-甲基化。特定 snoRNA 的失调与乳腺癌、前列腺癌、肺癌、脑癌、结直肠癌以及白血病和多发性骨髓瘤的风险增加有关，但 snoRNA 功能改变与癌症之间的机制联系尚不清楚。最近的发现预示着 snoRNA 领域的范式转变。首先，snoRNA 被证明具有大大扩展的潜在 RNA 靶标列表，包括 mRNA。其次，一些 snoRNA 指导的修饰被证明受到环境变化的调节。最后，假尿苷的存在被证明可以改变核糖体解释遗传密码的方式。总之，这些结果表明 snoRNA 通过指导 mRNA 的转录后修饰具有潜在广泛的调节功能。我们假设 snoRNA 通过一种涉及指导序列和靶标 RNA 之间宽松的特异性配对的机制来指导 mRNA 靶标的生理相关的假尿苷酰化。为了检验这一假设，我们开发了一种有效的方法，以单核苷酸精度（伪序列）在全基因组范围内绘制 snoRNA 指导的假尿苷酰化位点。我们的伪序列实验的初步结果提供了内源 mRNA 被伪尿苷酰化的第一个证据。因此，我们处于独特的有利位置来研究 snoRNA 功能的这种新范例。在目标 1 中，我们将使用 Pseudo-seq 确定 snoRNA 指导的假尿苷酰化的调控靶标，以响应营养缺乏、氧化应激和热休克。 snoRNA 酶催化成分 Cbf5/Dyskerin 中的突变体将用于确定哪些假尿苷是通过 snoRNA 依赖性机制形成的。完成这一目标将全面鉴定酵母中受调节的 snoRNA 依赖性修饰的 mRNA 靶标。目标 2 将使用计算和靶向遗传方法来剖析 snoRNA 调控的序列要求。这一目标的完成将有助于准确预测所有真核生物中的 snoRNA 靶标。最后，目标 3 将使用质谱来确定 mRNA 假尿嘧啶化的功能后果，以供核糖体解码。这一目标的完成有可能揭示遗传密码的重大重新布线。人类健康相关性：这项研究的结果将揭示 snoRNA 的新生物学功能，从而阐明 snoRNA 功能障碍导致癌症的潜在机制。我们相信，在未来，更好地了解 snoRNA 在细胞中的功能可能会更好地治疗与 snoRNA 基因功能扰动相关的癌症。