Computational Approaches for RNA StructureFunction Determination

RNA 结构功能测定的计算方法

• 批准号: 8348906
• 负责人: Bruce Shapiro
• 金额: $ 46.35万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8348906
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Acylation; Algorithms; Amino Acids; Bacterial Genome; Base Pairing; Beryllium; Binding; Biological; Biological Assay; Biology; Carrier Proteins; Catalysis; Categories; Cells; Cereals; Characteristics; Code; Complex; Computer Simulation; Computer software; Computing Methodologies; Conflict (Psychology); Coupling; Dengue; Dengue Virus; Development; Disease; Elements; Enhancers; Escherichia coli; Escherichia coli K12; Eukaryota; Frequencies; Functional RNA; Gene Expression; Gene Silencing; Genes; Genetic Enhancer Element; Genetic Programming; Genetic Transcription; Genome; Genomics; Higher Order Chromatin Structure; Human; Human Cell Line; Hydroxyl Radical; Individual; Knowledge; Length; Measurement; Measures; Mediating; Membrane; Messenger RNA; MicroRNAs; Modeling; Molecular Models; Nature; Nucleotides; Open Reading Frames; Pathway interactions; Positioning Attribute; Primer Extension; Property; Protein Binding; Protein Biosynthesis; Proteins; Publishing; RNA; RNA Decay; RNA Folding; RNA Sequences; RNA analysis; Regulation; Reporting; Research; Residual state; Response Elements; Ribosomes; Roentgen Rays; Series; Signal Transduction; Structure; Structure-Activity Relationship; System; Techniques; Transcriptional Regulation; Transfer RNA; Translating; Translation Process; Translations; Turnip - dietary; Untranslated RNA; Untranslated Regions; Variant; Viral; Virus; base; cancer cell; cis acting element; computer studies; computerized tools; data mining; drug development; indexing; medulloblastoma cell line; molecular modeling; protein degradation; research study; three dimensional structure; viral RNA; web site

Discovery and Characterization of a New Kind of Translational Enhancer 3' UTRs of cellular and viral mRNAs harbor elements that function in gene expression by enhancing translation using unknown mechanisms. To determine the function of these elements we used a simple model, the Turnip crinkle virus (TCV). TCV is translated in a cap-independent fashion and contains a 3' region that together with the 5' UTR synergistically enhances translation. We used MPGAfold and Structurelab to identify a series of hairpins and two pseudoknots that were confirmed genetically. Using this structural information with our 3D molecular modeling software, we predicted a structure that resembled a tRNA, the first internal tRNA-like structure found in nature. We then proposed that translational enhancement by the element might involve ribosome binding. The element was found to bind the 60S ribosomal subunit, the first such interaction with the large subunit discovered. It was biochemically determined that this tRNA-like element is a major part of a switch that converts the template from one that is translated to one that is replicated. We further investigated the formation of this unique translational enhancer utilizing a newly developed technique that combines Small Angle X-ray Scattering (SAXS) and Residual Dipolar Coupling (RDC) (see below). The results verified the basic model that had been predicted computationally and proved the efficacy of the technique for large RNAs, in addition to further characterizing this newly discovered translational enhancer element. This may open the door to the discovery of similar mechanisms in other genes. Characteristics that Determine Abundance of Two-Thirds of Proteins in a Human Cell Line Transcription, mRNA decay, translation, and protein degradation all contribute to steady state protein concentrations in multi-cellular eukaryotes. In this research, experimental measurements and computational studies were done to determine the absolute protein and mRNA abundances in cellular lysates from the human Daoy medulloblastoma cell line, and the properties that contributed to these abundances. Sequence features related to translation and protein degradation explained two-thirds of protein abundance variation. mRNA sequence lengths, amino acid properties, upstream open reading frames and secondary structures in the 5' untranslated region (UTR) showed the strongest individual correlations for protein concentrations. In a combined model, characteristics of the coding region and the 3'UTR explained a larger proportion of protein abundance variation than characteristics of the 5'UTR. Cis Acting Elements in the 3' UTR of Dengue Virus Over 50 million case of dengue fever are reported each year with 10% of these leading to severe forms of the disease. Using MPGAfold (our massively parallel genetic algorithm for RNA folding) we showed that the core region of the 3' untranslated region of dengue virus RNA can form two dumbell structures of unequal frequencies of occurence. It was experimentally shown that structural motifs formed from these dumbells are important for viral replication. In addition, it was shown that there is a cooperative synergy with both dumbells for translation. Thus, we showed that the cis-acting elements in the core region of dengue virus are require for both replication and optimal translation. Correlating SHAPE Signatures with 3D RNA Structures Selective 2-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is a relatively easy technique for the quantitative analysis of RNA secondary structure. In general, low SHAPE signal values are correlated with Watson-Crick base pairing, and high values indicate positions that are single-stranded within the RNA structure. The relationship of the measured SHAPE signal to structural properties such as non-Watson-Crick base pairing or the position of a nucleotide within an RNA double helix has thus far not been thoroughly investigated. In this research we presented results of SHAPE experiments performed on a set of seven RNAs with published 3D structures. We found that the RNA SHAPE signal depends on the type of base pairs a nucleotide is involved in; also we found a strong correlation between the SHAPE signal corresponding to a nucleotide and its position in an RNA double helix. Data Mining of Functional RNA Structures in Genomic Sequences The normal functions of genomes depend on the precise expression of messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs), such as microRNAs. These ncRNAs and functional RNA structures (FRSs) act as regulators or response elements for cellular factors, participate in transcription, post-transcriptional processing, and translation. In RNA-based regulation, the regulatory RNAs are often correlated with distinct higher-order structures. Computational simulations have indicated that a large number of FRSs are both significantly more structured and thermodynamically more stable. Various computational tools have been developed and the structural features of ncRNAs and FRSs have been determined. In this study we discuss our efforts in the computational discovery of structured features of ncRNAs and FRSs within complex genomes. Characterizing Structural Features for Small Regulatory RNAs in E. Coli Small regulatory RNAs are highly abundant noncoding RNAs (ncRNA) found in bacterial genomes. These small regulatory ncRNAs (sRNAs) can regulate the synthesis of proteins by mediating mRNA transcription, translation and stability. Furthermore, they also control the activity of specific proteins by binding to them. In this research, we describe a general computational approach for identifying the distinct structure of sRNAs in the Escherichia coli (E. coli) genomes by a quantitative measure that is the energy difference between the optimal structure folded from a sequence segment and its corresponding optimal restrained structure where all base pairings formed in the original optimal structure are excluded. Our results indicated that most of the known small ncRNAs in E. coli K12 have very high normalized scores with high statistical significance. These sRNAs have distinct well-ordered structures that are both thermodynamically stable and uniquely folded. CyloFold CyloFold is a new algorithm accessible via our webserver that predicts RNA secondary structure with pseudoknots. Pseudoknot prediction is unrestricted, thus permitting the formation of a multitude of pseudoknots with high degrees of complexity. A unique aspect of the algorithm is a coarse-grained mechanism that checks for steric feasibility of the chosen set of helices representing the structure. Helicical combinations that produce steric conflicts are eliminated from consideration in the predicted structure.

通过使用未知机制增强翻译，发现和表征了一种新型的转化增强剂3'UTR 3'UTR，它们在基因表达中起作用。为了确定这些元素的功能，我们使用了一个简单的模型，即萝卜皱纹病毒（TCV）。 TCV以盖帽独立的方式翻译，并包含一个3'区域，与5'UTR协同增强翻译相同。我们使用mpgafold和structurelab来识别一系列的发夹和两个伪造的伪造。使用这些结构信息与我们的3D分子建模软件，我们预测了类似于tRNA的结构，这是自然界中第一个类似于内部tRNA的结构。然后，我们提出，元素的翻译增强可能涉及核糖体结合。发现该元素结合了60年代的核糖体亚基，这是与发现的大亚基的第一个相互作用。在生化上确定的是，该tRNA样元素是开关的主要部分，该开关将模板从转换为复制的模板转换为一个模板。我们进一步研究了这种独特的翻译增强剂的形成，该技术利用新开发的技术结合了小角度X射线散射（SAXS）和残留的偶性偶联（RDC）（请参见下文）。结果验证了已经在计算上预测的基本模型，并证明了该技术对大型RNA的功效，此外还进一步表征了这种新发现的转化增强子元素。这可能打开了在其他基因中发现类似机制的大门。 确定人类细胞系转录，mRNA衰减，翻译和蛋白质降解中三分之二蛋白质的丰度的特征都有助于多细胞真核生物中的稳态蛋白质浓度。在这项研究中，进行了实验测量和计算研究，以确定人类daoy髓母细胞瘤细胞系中细胞裂解物中的绝对蛋白质和mRNA丰度，以及导致这些丰度的特性。与翻译和蛋白质降解有关的序列特征解释了三分之二的蛋白质丰度变化。 5'非翻译区域（UTR）中的mRNA序列长度，氨基酸性质，上游开放式阅读框和二级结构显示出蛋白质浓度最强的个体相关性。在组合模型中，与5'UTR的特征相比，编码区域和3'UTR的特征解释了蛋白质丰度变化的比例。每年据报道，登革热病毒的3'UTR中的顺式作用元素每年有10％的登革热病例，其中10％导致严重的疾病形式。使用MPGAFOLD（我们用于RNA折叠的大量平行遗传算法）我们表明，登革热病毒RNA的3'未翻译区域的核心区域可以形成两种出现频率的哑铃结构。经过实验表明，由这些哑铃形成的结构基序对于病毒复制很重要。此外，还表明两个哑铃都有合作的协同作用。因此，我们表明登革热病毒核心区域中的顺式作用元素既需要复制和最佳翻译。与3D RNA结构相关的形状特征选择性2-羟基酰基通过引物扩展分析（Shape）是对RNA二级结构进行定量分析的相对简单技术。通常，低形状的信号值与沃森 - 克里克基底配对相关，高值表示在RNA结构中单链的位置。迄今为止，尚未对测得的形状信号与结构特性（例如非沃特森 - 冰可碱基配对或核苷酸的位置）之间的关系。在这项研究中，我们介绍了对具有已发表3D结构的七个RNA进行的形状实验结果。我们发现RNA形状信号取决于基本对的类型，核苷酸参与了核苷酸。同样，我们发现与核苷酸对应的形状信号与其在RNA双螺旋中的位置之间存在很强的相关性。基因组序列中功能RNA结构的数据挖掘基因组的正常功能取决于Messenger RNA（mRNA）和非编码RNA（NCRNA）的精确表达，例如microRNA。这些NCRNA和功能性RNA结构（FRS）充当细胞因子的调节剂或响应元素，参与转录，转录后处理和翻译。在基于RNA的调控中，调节RNA通常与不同的高阶结构相关。计算模拟表明，大量FRS的结构更大，并且在热力学上更稳定。已经开发了各种计算工具，并确定了NCRNA和FRS的结构特征。在这项研究中，我们讨论了我们在复杂基因组中NCRNA和FRS的结构化特征的计算发现方面的努力。表征大肠杆菌中小调节性RNA的结构特征是细菌基因组中发现的高度丰富的非编码RNA（NCRNA）。这些小的调节性NCRNA（SRNA）可以通过介导mRNA转录，翻译和稳定性来调节蛋白质的合成。此外，它们还通过与它们结合来控制特定蛋白质的活性。在这项研究中，我们通过一种定量度量来描述一种通用计算方法，用于识别大肠杆菌（E. coli）基因组中SRNA的独特结构，该定量度量是从序列段折叠的最佳结构及其相应的最佳限制性结构之间的能量差，其中所有原始最佳结构中所有在原始最佳结构中形成的所有基本配对都被排除在外。我们的结果表明，大肠杆菌K12中的大多数已知小NCRNA具有很高的归一化分数，其统计学意义很高。这些SRNA具有不同有序的结构，它们在热力学上稳定且独特地折叠。 Cylofold Cylofold是可以通过我们的Web服务器访问的一种新算法，可以通过伪诺预测RNA二级结构。伪诺预测是不受限制的，因此允许形成具有高度复杂性的众多伪诺。该算法的独特方面是一种粗粒机制，该机制检查代表结构所选的一组螺旋的空间可行性。在预测结构中考虑的考虑因素消除了产生空间冲突的螺旋组合。