Regulation and Function of snoRNA Genes

描述（由申请人提供）：snoRNAs是高度保守的非编码rna，可指导其他rna的两种位点特异性转录后修饰-伪尿嘧啶化和2'- o -甲基化。特异性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，研究表明，snoRNAs具有极大扩展的潜在RNA靶标列表，包括mrna。其次，一些由snorna引导的修饰被证明受到环境变化的调节。最后，假尿嘧啶的存在被证明改变了核糖体解释遗传密码的方式。总之，这些结果表明，通过指导mrna的转录后修饰，snoRNAs可能具有广泛的调节功能。我们假设，snoRNAs通过一种涉及引导序列和靶RNA之间放松特异性配对的机制，直接生理上相关的mRNA假尿嘧啶化靶标。为了验证这一假设，我们开发了一种有效的方法，以单核苷酸精度在全基因组范围内绘制snorna导向的伪尿嘧啶化位点（Pseudo-seq）。伪序列实验的初步结果提供了内源性mrna被伪尿嘧啶化的第一个证据。因此，我们具有独特的优势来研究snoRNA功能的这种新范式。在Aim 1中，我们将使用Pseudo-seq识别在营养剥夺、氧化应激和热休克下，由snorna引导的假尿嘧啶化的调控靶标。snoRNA酶的催化组分Cbf5/Dyskerin的突变体将用于确定哪些假尿嘧啶是通过snoRNA依赖机制形成的。这一目标的完成将全面鉴定酵母中受调控的snorna依赖性修饰的mRNA靶标。目的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靶点。最后，Aim 3将使用质谱来确定mRNA假尿嘧啶化对核糖体解码的功能后果。这一目标的完成有可能揭示遗传密码的大量重新布线。人类健康相关性：本研究结果将揭示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，更好地了解细胞中snoRNA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。

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.