Computational Approaches for RNA StructureFunction Determination

RNA 结构功能测定的计算方法

• 批准号: 8157206
• 负责人: Bruce Shapiro
• 金额: $ 49.58万
• 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/8157206
• 关键词: Algorithms; Amino Acids; Binding; Biological; CCR; Cell Maintenance; Characteristics; Collaborations; Data; Development; Elements; Equilibrium; Gene Expression; Gene Targeting; Genes; Genetic Enhancer Element; Glioblastoma; Guanine + Cytosine Composition; Hepatitis B Virus; Hepatitis Delta Virus; Higher Order Chromatin Structure; Human; Individual; Initiator Codon; Institutes; Length; Link; Liver diseases; Malignant - descriptor; Malignant neoplasm of liver; Maryland; Measurement; Methodology; MicroRNAs; Molecular Conformation; Molecular Models; Nature; New Jersey; Pathway interactions; Peptides; Phase; Process; Proteins; RNA; RNA Sequences; Research; Residual state; Ribosomes; Series; Shapes; Terminator Codon; Transcriptional Regulation; Variant; X-Ray Crystallography; adenosine deaminase; base; cancer cell; data mining; improved; mRNA Decay; outcome forecast; tumorigenesis; web site

Applications RNA Structure prediction and analysis HDV MPGAfold and StructureLab were applied in a study of the Hepatitis Delta virus (HDV). HDV is a virus associated with the Hepatitis B virus (HBV). HDV with HBV increases the severity of liver disease and enhances the likelihood of developing liver cancer. HDV produces one protein, the hepatitis delta antigen, which has two forms, the short and the long form. We showed, with the use of MPGAfold, that the Ecuadorian strain (ES) attains two secondary structures that are crucial for functionality. The HDV RNA is edited when it attains a branched conformation, changing a stop codon into a tryptophan. Later, the virus changes into a linear form which is necessary for replication, leading to the translation of a longer peptide which inhibits viral synthesis. At times the RNA bypasses the branched form and attains the linear replication form, avoiding editing, resulting in a shorter peptide required for HDV replication. Recently, MPGAfold indicated that the Peruvian strain (PS) had different folding characteristics than ES. ES attained the editing structure more readily. Our collaborator John Casey verified this with experiments and showed that ES binds to its editing protein adenosine deaminase less efficiently than PS. These results showed that HDV strains maintain a delicate balance between the formation of the editing and replication states. 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. In collaboration with Professor Anne Simon, from the University of Maryland, we are deciphering the function of this 3' element. 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. In collaboration with Yun-Xing Wang, NCI-Frederick, 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. eIF4E We determined the properties associated with specific mRNAs that are translationally enhanced due to the overexpression of oncogenic eIF4E in cancer cells. We showed that structuredness in the 5 UTR was not the sole determinant of up-regulation. We showed that up-regulated mRNAs have on average shorter 3 UTRs, higher G+C content and slightly more RNA secondary structure before the start codon and around the stop codon. Another feature is the apparent diminution of binding sites for microRNAs known to be tumor suppressors for mRNAs that are highly responsive to increased eIF4E concentration. A machine classifier was tested which distinguishes between these cases. Characteristics that Determine Abundance 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 collaboration with Luiz Penalva from the University of Texas, 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. Musashi Mushashi1(Msi1) is a highly conserved RNA binding protein with pivotal functions in stem cell maintenance and development of the nervous system. There is evidence that links Msi1 to tumor formation; its expression has been observed in a variety of tumor types and high levels of expression have been correlated with poor prognosis in glioblastomas and astrocytomas. A high-throughput approach was used by our collaborator,Luiz Penalva at the University of Texas, to identify a group of target mRNAs to elucidate their participation in stem cell maintenance, cell differentiation and tumorigenesis. We applied a computational data mining approach to find the regulatory signal and structural motif in the 3' UTR of these Msi1 targeted genes. Results from experimental and computational data indicated that the Msi1 binding ability depends on multiple factors including closely correlated conserved binding sequences and an associated RNA structural motif detected in the 3'UTRs. RNA Structure Prediction and Analysis Software: 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. Pseudo energy minimization Simulation algorithms that are based on thermodynamic processes often minimize the free energy of folding of single RNA sequences to predict their secondary structures. The additional use of covariance scores derived from multiple sequence alignments can improve the accuracy of these predictions. We developed with Jason Wang at the New Jersey Institute of Technology, an algorithm, RSpredict, that predicts the consensus secondary structure of a set of aligned sequences that combines the principles of dynamic programming with covariation scores. Combining NMR and SAXS The determination of large 3D RNA structures by NMR, X-ray crystallography or other experimental techniques has been a very difficult problem. Our group with Yun-Xing Wang's group in CCR, has developed a methodology that combines techniques from NMR and SAXS to determine the global architecture of large RNAs consisting mostly of A-form helices. The determination of the orientation and the rotation of helices around their helical axes and the relative global positions of the helices can be used to determine structure. SAXS is used to determine the envelop of the shape of the molecule and RDC is used to determine the relative orientations and rotational phases of the helices.

应用 RNA 结构预测和分析 HDV MPGAfold 和 StructureLab 应用于丁型肝炎病毒 (HDV) 的研究。 HDV 是一种与乙型肝炎病毒 (HBV) 相关的病毒。丁型肝炎病毒（HDV）合并乙型肝炎病毒（HDV）会增加肝脏疾病的严重程度，并增加患肝癌的可能性。丁型肝炎病毒产生一种蛋白质，即丁型肝炎抗原，它有两种形式：短形式和长形式。我们表明，通过使用 MPGAfold，厄瓜多尔菌株 (ES) 获得了两个对功能至关重要的二级结构。 HDV RNA 在达到分支构象时会被编辑，将终止密码子变为色氨酸。随后，病毒转变成复制所必需的线性形式，导致翻译出更长的肽，从而抑制病毒合成。有时，RNA 绕过分支形式并获得线性复制形式，避免编辑，从而产生 HDV 复制所需的较短肽。最近，MPGAfold 表明秘鲁菌株（PS）具有与 ES 不同的折叠特征。 ES更容易获得编辑结构。我们的合作者 John Casey 通过实验验证了这一点，并表明 ES 与其编辑蛋白腺苷脱氨酶的结合效率低于 PS。这些结果表明 HDV 菌株在编辑状态和复制状态的形成之间保持着微妙的平衡。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。我们与马里兰大学的 Anne Simon 教授合作，正在破译这个 3' 元件的功能。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。我们与 NCI-Frederick 的 Yun-Xing Wang 合作，利用新开发的技术，结合小角 X 射线散射 (SAXS) 和残余偶极耦合 (RDC)（见下文），进一步研究了这种独特翻译增强子的形成。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。 eIF4E 我们确定了与特定 mRNA 相关的特性，这些 mRNA 由于癌细胞中致癌 eIF4E 的过度表达而翻译增强。我们表明，5 UTR 的结构性并不是上调的唯一决定因素。我们发现上调的 mRNA 平均具有更短的 3 个 UTR、更高的 G+C 含量以及在起始密码子之前和终止密码子周围稍多的 RNA 二级结构。另一个特征是 microRNA 的结合位点明显减少，这些 microRNA 是对 eIF4E 浓度增加高度敏感的 mRNA 的肿瘤抑制因子。测试了区分这些情况的机器分类器。决定人类细胞系中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。与德克萨斯大学的 Luiz Penalva 合作，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。 Musashi Mushashi1(Msi1) 是一种高度保守的 RNA 结合蛋白，在干细胞维持和神经系统发育中具有关键功能。有证据表明 Msi1 与肿瘤形成有关；已在多种肿瘤类型中观察到其表达，并且高水平表达与胶质母细胞瘤和星形细胞瘤的不良预后相关。我们的合作者德克萨斯大学的 Luiz Penalva 使用高通量方法鉴定了一组目标 mRNA，以阐明它们在干细胞维持、细胞分化和肿瘤发生中的参与。我们应用计算数据挖掘方法来寻找这些 Msi1 靶基因 3' UTR 中的调控信号和结构基序。实验和计算数据的结果表明，Msi1 结合能力取决于多种因素，包括密切相关的保守结合序列和在 3'UTR 中检测到的相关 RNA 结构基序。 RNA 结构预测和分析软件：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。伪能量最小化 基于热力学过程的模拟算法通常会最小化单个 RNA 序列的折叠自由能，以预测其二级结构。额外使用从多个序列比对得出的协方差分数可以提高这些预测的准确性。我们与新泽西理工学院的 Jason Wang 一起开发了一种算法 RSpredict，该算法结合了动态规划和协变分数的原理，可以预测一组比对序列的一致二级结构。 NMR 和 SAXS 的结合通过 NMR、X 射线晶体学或其他实验技术测定大型 3D RNA 结构一直是一个非常困难的问题。我们的团队与 CCR 的 Yun-Xing Wang 团队开发了一种方法，结合 NMR 和 SAXS 技术来确定主要由 A 型螺旋组成的大 RNA 的整体结构。螺旋绕其螺旋轴的方向和旋转以及螺旋的相对整体位置的确定可用于确定结构。 SAXS 用于确定分子形状的包络，RDC 用于确定螺旋的相对方向和旋转相位。