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.

一种新的细胞和病毒mrna的翻译增强子3' UTRs的发现和表征通过未知机制增强翻译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，萝卜皱病毒（TCV）。TCV以帽独立的方式翻译，包含一个3‘区域，与5’ UTR一起协同增强翻译。我们使用mpgfold和Structurelab鉴定了一系列的发夹和两个假结，这些发夹和假结在遗传学上得到了证实。利用这些结构信息和我们的3D分子建模软件，我们预测了一个类似tRNA的结构，这是自然界中发现的第一个内部tRNA结构。然后我们提出该元件的翻译增强可能涉及核糖体结合。该元件被发现与60S核糖体亚基结合，这是首次发现与大亚基相互作用。从生物化学角度确定，这种trna样元素是一个开关的主要部分，它将模板从一个被翻译的转换为一个被复制的。我们利用一种结合了小角x射线散射（SAXS）和残余偶极耦合（RDC）的新技术进一步研究了这种独特的平移增强子的形成（见下文）。结果验证了计算预测的基本模型，证明了该技术对大rna的有效性，并进一步表征了这一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了大门。转录、mRNA衰变、翻译和蛋白质降解都有助于多细胞真核生物稳定的蛋白质浓度。在本研究中，通过实验测量和计算研究，确定了人髓母细胞瘤细胞系细胞裂解物中蛋白质和mRNA的绝对丰度，以及促成这些丰度的特性。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化。mRNA序列长度、氨基酸性质、上游开放阅读框和5'非翻译区（UTR）二级结构与蛋白质浓度的个体相关性最强。在一个组合模型中，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化。每年报告的登革热病例超过5000万例，其中10%导致严重形式的登革热。利用MPGAfold（我们用于RNA折叠的大规模并行遗传算法），我们发现登革热病毒RNA的3'非翻译区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，由这些哑铃形成的结构基序对病毒复制很重要。此外，研究还表明，两个哑铃在翻译过程中存在协同效应。因此，我们证明了登革热病毒核心区的顺式作用元件是复制和最佳翻译所必需的。通过引物延伸分析选择性2-羟基酰化（SHAPE）是一种相对简单的RNA二级结构定量分析技术。一般来说，低的SHAPE信号值与沃森-克里克碱基配对相关，高的值表示RNA结构中单链的位置。测量到的形状信号与结构特性的关系，如非沃森-克里克碱基配对或RNA双螺旋内核苷酸的位置，迄今尚未得到彻底的研究。在这项研究中，我们展示了对一组具有已发表的3D结构的7种rna进行的SHAPE实验结果。我们发现RNA形状信号取决于核苷酸所参与的碱基对类型；我们还发现，核苷酸对应的形状信号与其在RNA双螺旋结构中的位置之间存在很强的相关性。基因组的正常功能依赖于信使RNA （mrna）和非编码RNA (ncrna)（如microrna）的精确表达。这些ncrna和功能性RNA结构（FRSs）作为细胞因子的调节因子或应答元件，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，大量的frs结构明显更强，热力学更稳定。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计算发现ncrna和FRSs结构特征方面的努力。小调控rna是在细菌基因组中大量存在的非编码rna （ncRNA）。这些小调控ncRNAs （sRNAs）可以通过介导mRNA的转录、翻译和稳定性来调控蛋白质的合成。此外，它们还通过与特定蛋白质的结合来控制它们的活性。在这项研究中，我们描述了一种通用的计算方法，用于识别大肠杆菌（E. coli）基因组中sRNAs的独特结构，通过定量测量从序列片段折叠的最佳结构与其相应的最佳约束结构之间的能量差，其中排除了原始最佳结构中形成的所有碱基对。我们的研究结果表明，大肠杆菌K12中大多数已知的小ncrna具有非常高的归一化得分，具有很高的统计学意义。这些sRNAs具有独特的有序结构，既具有热力学稳定性，又具有独特的折叠。CyloFold是一个新的算法，可通过我们的web服务器访问，预测RNA二级结构与假结。假结预测是不受限制的，因此允许形成具有高度复杂性的大量假结。该算法的一个独特之处在于它采用了一种粗粒度机制，用于检查所选的表示结构的螺旋集的立体可行性。在预测结构中不考虑产生空间冲突的螺旋组合。