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.

一种新型翻译增强子的发现和表征细胞和病毒mrna的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。为了确定这些元素的功能，我们之前使用了萝卜皱病毒（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。我们最近在豌豆代花叶病毒中发现了另一种这种类型的元件，然而，这种翻译增强元件以某种不同的方式起作用。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，以及实验方法来证明该元素是t形的，这让人想起TCV与tRNA的相似性。我们发现它与核糖体结合并参与长距离RNA-RNA相互作用。它的功能提示了RNA 5‘/3’环化的一种手段，因此是一种增强翻译的机制。这些新元素的存在似乎暗示了在真核生物中可能发现的替代翻译机制。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。登革热病毒3' UTR中的顺式作用因子每年报告的登革热病例超过5000万例，其中10%导致疾病的严重形式。利用我们的RNA折叠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2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。结果表明，两个哑铃在翻译过程中具有协同效应。因此，登革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）是一种相对简单的定量分析RNA二级结构的技术。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（如非沃森-克里克碱基配对）之间的关系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结构的七个rna进行的SHAPE实验的结果。我们发现RNA形状信号取决于核苷酸参与的碱基对的类型，它的堆叠能力和它的环境。这对于改进RNA结构预测的计算/实验方法具有重要意义。离子和侧翼碱基在HIV-1二聚化中的作用实验表明，HIV-1接吻环形成的特征取决于病毒的亚型。研究表明，b亚型单体在高盐浓度或镁离子存在下二聚化，而a亚型单体只与镁离子结合在G273或G274的磷酸基上二聚化，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明显变形，发夹环中与二聚化有关的碱基向内转动。在高盐浓度下，b亚型单体保持有利于二聚化的形状，而a亚型单体仍表现出明显的变形。b亚型的侧翼碱基有助于稳定构象，而a亚型的侧翼碱基G273引起变形。然而，当镁离子存在并与G273或G274磷酸基团结合时，碱G273保持稳定环二聚化的构象。这些结果对于理解HIV-1病毒生命周期的机制非常重要。通过组合特征挖掘和促进microrna分类Pre-miRNA是由来自前体分子的约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。在扫描基因组时，很难将假阳性的pre- mirna与真实的区分开来。本研究提出了一种鉴定和分类pre- mirna的新方法。采用组合特征挖掘的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。然后应用增强算法进一步提高精度。结果表明，与以往的方法相比，有显著的改进。基因组序列中功能性RNA结构的数据挖掘基因组的正常功能依赖于mrna和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具有明显的结构和热力学稳定性。已经开发了各种计算工具，并确定了ncrna和frs的结构特征。我们报告了我们在复杂基因组中计算发现ncrna和frs结构特征方面的努力。发现两个RNA序列中的共同折叠模式开发了一种高效的动态规划算法，该算法使用有序标记树来发现给定2个RNA序列中最大的共同RNA亚结构。该算法还可用于发现RNA二级结构的重复区域。大肠杆菌小调控rna是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具有较高的统计结构显著性，并且热力学稳定。