Regulation and Function of snoRNA Genes

snoRNA基因的调控和功能

• 批准号: 9495443
• 负责人: Wendy Victoria Gilbert
• 金额: $ 31.83万
• 依托单位: YALE UNIVERSITY
• 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/9495443
• 关键词: Affect; Amino Acids; Biological Process; Biology; Cancer Patient; Cells; Cellular Stress; Codon Nucleotides; Colorectal Cancer; Data; Enzymes; Eukaryota; Functional disorder; Future; Genes; Genetic Code; Genetic Transcription; 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; Mutation; Nucleotides; Organism; Oxidative Stress; Pattern; Physiological; Positioning Attribute; Pseudouridine; RNA; Regulation; Ribosomal RNA; Ribosomes; Risk; Sense Codon; Site; Small Nuclear RNA; Small Nucleolar RNA; Specificity; Techniques; Terminator Codon; Testing; Therapeutic; Time; Translating; Translations; Treatment-Related Cancer; United States; Untranslated RNA; Validation; Woman; Work; Yeasts; cancer therapy; design; environmental change; experimental study; genetic approach; genome-wide; killings; leukemia; malignant breast neoplasm; men; mutant; nutrient deprivation; public health relevance; 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，其指导其他RNA的两种类型的位点特异性转录后修饰-假尿苷酸化和2 '-O-甲基化。特定snoRNA的失调与乳腺癌、前列腺癌、肺癌、脑癌和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被证明具有极大扩展的潜在RNA靶点列表，包括mRNA。第二，一些snoRNA定向修饰被证明受环境变化的调节。最后，假尿苷的存在被证明可以改变核糖体对遗传密码的解释。总之，这些结果表明snoRNA通过指导mRNA的转录后修饰具有潜在的广泛调节功能。我们假设snoRNA通过一种机制指导mRNA靶点的生理相关假尿苷化，该机制涉及指导序列和靶RNA之间的松弛特异性配对。为了验证这一假设，我们已经开发了一种有效的方法，用于以单核苷酸精确度（Pseudo-seq）在全基因组范围内定位snoRNA指导的假尿苷酸化位点。我们来自伪序列实验的初步结果提供了内源性mRNA被假尿苷化的第一个证据。因此，我们处于独特的有利地位，以研究snoRNA功能的这种新范式。在目标1中，我们将使用Pseudo-seq鉴定响应于营养剥夺、氧化应激和热休克的snoRNA指导的假尿苷化的调节靶标。snoRNA酶的催化组分中的突变体Cbf 5/Dyskerin将用于确定哪些假尿苷是由snoRNA依赖性机制形成的。这一目标的完成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将使用计算和有针对性的遗传方法来剖析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假尿苷化对核糖体解码的功能后果。完成这一目标有可能揭示遗传密码的实质性重组。人类健康相关性：这项研究的结果将揭示snoRNA的新的生物学功能，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导致更好地治疗与snoRNA基因功能干扰相关的癌症。