Computational Approaches for RNA StructureFunction Determination

RNA 结构功能测定的计算方法

• 批准号: 8552600
• 负责人: Bruce Shapiro
• 金额: $ 45.16万
• 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/8552600
• 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; Characteristics; Classification; Code; Complex; Computer Simulation; Computer software; Computing Methodologies; Cyclization; Dengue; Dengue Virus; Development; Dimerization; Disease; Elements; Escherichia coli; Escherichia coli K12; Eukaryota; Frequencies; Functional RNA; Gene Expression; Gene Silencing; Genetic Enhancer Element; Genetic Programming; Genetic Transcription; Genome; Genome Scan; Genomics; HIV; HIV-1; Higher Order Chromatin Structure; Human; Hydroxyl Radical; Individual; Ions; Knowledge; Label; Length; Life Cycle Stages; Machine Learning; Measures; Mediating; Membrane; Messenger RNA; Methods; MicroRNAs; Mining; Modeling; Molecular Conformation; Nucleotides; Open Reading Frames; Organism; Pathway interactions; Pattern; Positioning Attribute; Primer Extension; Property; Protein Binding; Protein Biosynthesis; Proteins; Publishing; RNA; RNA Folding; RNA Sequences; RNA analysis; Regulation; Reporting; Research; Response Elements; Ribosomes; Role; Shapes; Signal Transduction; Sodium Chloride; Structure; Structure-Activity Relationship; System; Techniques; Training Support; Transcriptional Regulation; Transfer RNA; Translating; Translation Process; Translations; Trees; Turnip - dietary; Untranslated RNA; Untranslated Regions; Variant; Viral; Virus; base; cancer cell; cis acting element; combinatorial; computer studies; computerized tools; data mining; drug development; improved; indexing; inorganic phosphate; mRNA Decay; mRNA Expression; magnesium ion; medulloblastoma cell line; monomer; novel; pea enation mosaic virus; pre-miRNA; programs; protein degradation; research study; stem; three dimensional structure; viral RNA; web site

Discovery and Characterization of a Novel Translational Enhancers3' 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 previously used 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 recently discovered another element of this type in the Pea enation mosaic virus, however, this translational enhancer element act in a somewhat different way. We used a set of computational tools, including RNA2D3D for 3D RNA modeling, developed in our lab, and experimental methods to show that this element is T-shaped, which is reminiscent of TCV's similarity to tRNA. We showed that it binds ribosomes and engages in a long range RNA-RNA interaction. Its functionality suggests a means for RNA 5'/3' cyclization and thus a mechanism for translational enhancement. It appears that the existence of these novel elements is suggesting alternate translational mechanisms that may be found in eukaryotic organisms.Characteristics that Determine Abundance of Two-Thirds of Proteins in a Human Cell LineTranscription, mRNA decay, translation, and protein degradation all contribute to steady state protein concentrations in multi-cellular eukaryotes. In this research, experimental 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 VirusOver 50 million case of dengue fever are reported each year with 10% leading to severe forms of the disease. Using 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 2 dumbell structures of unequal frequencies of occurrence. It was experimentally shown that structural motifs formed from these dumbells are important for viral replication. Also it was shown that there is a cooperative synergy with both dumbells for translation. Thus, the cis-acting elements in the core region of dengue virus are required for both replication and optimal translation.Correlating SHAPE Signatures with 3D RNA StructuresSelective 2-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is a relatively easy technique for the quantitative analysis of RNA secondary structure. 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 has thus far not been thoroughly investigated. In this research we present 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, its ability to stack and its context. This is important for improving computational/experimental methods for RNA structure prediction.The Role of Ions and Flanking Bases in the Dimerization of HIV-1Experimentally it has been shown that the characterization of HIV-1 kissing loop formation differs depending on the subtype of the virus. It has been shown that subtype-B monomers dimerize at high salt concentrations or in the presence of magnesium ions while subtype-A monomers will only dimerize with a magnesium ion bound to the flanking G273 phosphate group or the phosphate group of G274 regardless of the salt concentration. We found using computer simulations that at low concentrations both types of monomer hairpin loops were significantly deformed and the bases in the hairpin loop that are associated with dimerization were turned inward. At high salt concentrations the subtype-B monomer maintained a shape conducive to dimerization, while subtype-A still showed significant deformations. Also the flanking bases in subtype-B helped to stabilize the conformation while the flanking base G273 in subtype-A caused deformation. However, when magnesium ions were present and bound to the G273 or G274 phosphate groups, base G273 maintained a conformation that stabilized the loop for dimerization. These results are important for understanding the mechanisms that are involved in the HIV-1 virus life cycle.Classifying Pre-miRNA via Combinatorial Feature Mining and BoostingMicroRNAs are non-coding RNAs consisting of about 22 nucleotides that are derived from precursor molecules. These precursors usually fold into stem-loop hairpin structures. When scanning genomes it is difficult to distinguish false positive pre-miRNAs from the real thing. In this research a new method was developed for identifying and classifying pre-miRNAs. A combinatorial feature mining approach was used to discover a good set of features. These feature sets were then used to train support vector machines to obtain classification models. A boosting algorithm was then applied to further enhance the accuracy. Results indicate significant improvement over previous methods.Data Mining of Functional RNA Structures in Genomic SequencesThe normal functions of genomes depend on the precise expression of mRNAs and 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 indicated that a large number of FRSs are significantly more structured and thermodynamically stable. Various computational tools have been developed and the structural features of ncRNAs and FRSs have been determined. We report our efforts in the computational discovery of structured features of ncRNAs and FRSs within complex genomes.Discovering Common Folding Patterns in two RNA SequencesAn efficient dynamic programming algorithm was developed that uses ordered labeled trees for discovering the largest common RNA substructures given 2 RNA sequences. This algorithm can also be used to discover repeated regions of RNA secondary structure.Characterizing Structural Features for Small Regulatory RNAs in E. ColiSmall 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 high statistical structural significance and are thermodynamically stable.

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而在基因表达中起作用的元件。为了确定这些元件的功能，我们之前使用了芜菁皱纹病毒（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。我们最近在豌豆花叶病毒中发现了这种类型的另一种元件，然而，这种翻译增强子元件的作用方式有些不同。我们使用了一套计算工具，包括我们实验室开发的用于 3D RNA 建模的 RNA2D3D，以及实验方法来表明该元素是 T 形的，这让人想起 TCV 与 tRNA 的相似性。我们证明它与核糖体结合并参与长范围的 RNA-RNA 相互作用。它的功能表明了一种 RNA 5'/3' 环化方法，从而提供了一种翻译增强机制。这些新元素的存在似乎暗示了真核生物中可能存在的替代翻译机制。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 万登革热病例，其中 10% 导致严重的疾病形式。使用我们的大规模并行遗传算法进行RNA折叠，我们发现登革热病毒RNA 3'非翻译区的核心区域可以形成2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。还表明，两个哑铃在翻译方面具有协同作用。因此，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 结构相关联通过引物延伸 (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。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，测量的 SHAPE 信号与非沃森-克里克碱基配对等结构特性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、其堆叠能力及其上下文。这对于改进 RNA 结构预测的计算/实验方法非常重要。离子和侧翼碱基在 HIV-1 二聚化中的作用实验表明，HIV-1 接吻环形成的特征因病毒亚型而异。研究表明，B 亚型单体在高盐浓度或存在镁离子的情况下会发生二聚，而 A 亚型单体只会与与侧翼 G273 磷酸基团或 G274 磷酸基团结合的镁离子发生二聚，而与盐浓度无关。我们通过计算机模拟发现，在低浓度下，两种类型的单体发夹环都显着变形，并且发夹环中与二聚化相关的碱基向内转。在高盐浓度下，B 亚型单体保持有利于二聚化的形状，而 A 亚型仍然表现出明显的变形。此外，B 亚型中的侧翼碱基有助于稳定构象，而 A 亚型中的侧翼碱基 G273 会导致变形。然而，当镁离子存在并与 G273 或 G274 磷酸基团结合时，碱基 G273 保持稳定环二聚化的构象。这些结果对于理解 HIV-1 病毒生命周期中涉及的机制非常重要。通过组合特征挖掘和增强对 Pre-miRNA 进行分类 MicroRNA 是由源自前体分子的约 22 个核苷酸组成的非编码 RNA。这些前体通常折叠成茎环发夹结构。扫描基因组时，很难区分假阳性 pre-miRNA 和真实的 pre-miRNA。在这项研究中，开发了一种用于识别和分类 pre-miRNA 的新方法。使用组合特征挖掘方法来发现一组好的特征。然后使用这些特征集来训练支持向量机以获得分类模型。然后应用增强算法来进一步提高准确性。结果表明比以前的方法有显着的改进。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mRNA和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且热力学稳定。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。我们报告了我们在计算发现复杂基因组中 ncRNA 和 FRS 的结构化特征方面所做的努力。发现两个 RNA 序列中的常见折叠模式开发了一种高效的动态编程算法，该算法使用有序标记树来发现给定 2 个 RNA 序列的最大常见 RNA 子结构。该算法还可用于发现 RNA 二级结构的重复区域。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限制结构之间的能量差，其中排除了原始最佳结构中形成的所有碱基配对。我们的结果表明，大肠杆菌 K12 中大多数已知的小 ncRNA 具有较高的统计结构显着性并且热力学稳定。