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.

新型翻译增强子的发现和表征 细胞和病毒 mRNA 的 3' UTR 含有通过使用未知机制增强翻译来在基因表达中发挥作用的元件。为了确定这些元素的功能，我们使用了一个简单的模型，即芜菁皱纹病毒 (TCV)。 TCV 以不依赖帽子的方式进行翻译，并包含一个 3' 区域，与 5' UTR 一起协同增强翻译。我们使用 MPGAfold 和 Structurelab 识别了一系列发夹和两个经基因证实的假结。通过我们的 3D 分子建模软件使用这些结构信息，我们预测了一种类似于 tRNA 的结构，这是自然界中发现的第一个内部类似 tRNA 的结构。然后我们提出该元素的翻译增强可能涉及核糖体结合。该元素被发现与 60S 核糖体亚基结合，这是首次发现与大亚基的相互作用。生物化学证实，这种类似 tRNA 的元件是将模板从翻译模板转换为复制模板的开关的主要部分。我们利用新开发的技术进一步研究了这种独特的平移增强子的形成，该技术结合了小角 X 射线散射 (SAXS) 和残余偶极耦合 (RDC)（见下文）。结果验证了计算预测的基本模型，并证明了该技术对大RNA的有效性，此外还进一步表征了这种新发现的翻译增强子元件。这可能为在其他基因中发现类似机制打开大门。 决定人类细胞系中三分之二蛋白质丰度的特征转录、mRNA 衰变、翻译和蛋白质降解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 髓母细胞瘤细胞系细胞裂解物中蛋白质和 mRNA 的绝对丰度，以及导致这些丰度的特性。与翻译和蛋白质降解相关的序列特征解释了三分之二的蛋白质丰度变异。 mRNA 序列长度、氨基酸特性、上游开放阅读框和 5' 非翻译区 (UTR) 的二级结构显示出与蛋白质浓度最强的个体相关性。在组合模型中，编码区和 3'UTR 的特征比 5'UTR 的特征解释了更大比例的蛋白质丰度变异。登革热病毒 3' UTR 中的顺式作用元件 每年报告超过 5000 万例登革热病例，其中 10% 会导致严重的疾病。使用MPGAfold（我们用于RNA折叠的大规模并行遗传算法），我们证明登革热病毒RNA 3'非翻译区的核心区域可以形成两个出现频率不等的哑铃结构。实验表明，这些哑铃形成的结构基序对于病毒复制很重要。此外，结果表明，两个哑铃在翻译方面存在协同作用。因此，我们表明登革热病毒核心区域的顺式作用元件是复制和最佳翻译所必需的。将 SHAPE 特征与 3D RNA 结构关联起来 通过引物延伸 (SHAPE) 分析选择性 2-羟基酰化是一种相对简单的 RNA 二级结构定量分析技术。一般来说，低 SHAPE 信号值与 Watson-Crick 碱基配对相关，高值表示 RNA 结构内的单链位置。迄今为止，测得的 SHAPE 信号与结构特性（例如非 Watson-Crick 碱基配对或 RNA 双螺旋内核苷酸的位置）的关系尚未得到彻底研究。在这项研究中，我们展示了对一组七个已发布 3D 结构的 RNA 进行的 SHAPE 实验结果。我们发现 RNA SHAPE 信号取决于核苷酸所涉及的碱基对的类型；我们还发现对应于核苷酸的 SHAPE 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信使RNA（mRNA）和非编码RNA（ncRNA）（例如microRNA）的精确表达。这些 ncRNA 和功能性 RNA 结构 (FRS) 作为细胞因子的调节子或响应元件，参与转录、转录后加工和翻译。在基于 RNA 的调控中，调控 RNA 通常与不同的高阶结构相关。计算模拟表明，大量 FRS 的结构明显更加结构化，并且热力学更加稳定。各种计算工具已经被开发出来，并且 ncRNA 和 FRS 的结构特征已经确定。在这项研究中，我们讨论了我们在复杂基因组内 ncRNA 和 FRS 的结构化特征的计算发现方面所做的努力。表征大肠杆菌中小调节 RNA 的结构特征 小调节 RNA 是在细菌基因组中发现的高度丰富的非编码 RNA (ncRNA)。这些小型调节性 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，它们还通过与特定蛋白质结合来控制其活性。在这项研究中，我们描述了一种通用计算方法，用于通过定量测量来识别大肠杆菌 (E. coli) 基因组中 sRNA 的独特结构，即从序列片段折叠的最佳结构与其相应的最佳约束之间的能量差。排除在原始最佳结构中形成的所有碱基配对的结构。我们的结果表明，大肠杆菌 K12 中大多数已知的小 ncRNA 具有非常高的标准化分数，具有很高的统计显着性。这些 sRNA 具有独特的有序结构，既热力学稳定又独特折叠。 CyloFold CyloFold 是一种可通过我们的网络服务器访问的新算法，可通过假结预测 RNA 二级结构。假结预测不受限制，因此允许形成大量具有高度复杂性的假结。该算法的一个独特方面是粗粒度机制，用于检查代表结构的所选螺旋集的空间可行性。产生空间冲突的螺旋组合在预测结构中被排除在外。