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mpgfold和StructureLab对丁型肝炎病毒（HDV）进行了RNA结构预测和分析。HDV是一种与乙型肝炎病毒（HBV）相关的病毒。HDV合并HBV会增加肝脏疾病的严重程度，并增加发生肝癌的可能性。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。我们发现，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。当HDV RNA达到分支构象时，它被编辑，将停止密码子变成色氨酸。随后，病毒转变为复制所必需的线性形式，导致翻译更长的抑制病毒合成的肽。有时，RNA绕过分支形式，达到线性复制形式，避免了编辑，导致HDV复制所需的肽更短。最近MPGAfold显示秘鲁菌株（PS）与ES具有不同的折叠特征。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了微妙的平衡。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。与马里兰大学的安妮·西蒙教授合作，我们正在破译这个3'元素的功能。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。在与NCI-Frederick的合作中，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。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的过表达而被翻译增强。我们发现5utr的结构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，并且在开始密码子之前和停止密码子周围的RNA二级结构略多。另一个特征是microrna的结合位点明显减少，而microrna是对eIF4E浓度升高高度敏感的mrna的肿瘤抑制因子。测试了一种机器分类器来区分这些情况。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。与来自德克萨斯大学的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。Musashi Mushashi1（Msi1）是一种高度保守的RNA结合蛋白，在神经系统的干细胞维持和发育中具有关键作用。有证据表明Msi1与肿瘤形成有关；它在多种肿瘤类型中均有表达，在胶质母细胞瘤和星形细胞瘤中高水平表达与预后不良相关。我们的合作者，德克萨斯大学的Luiz Penalva使用了一种高通量方法来鉴定一组靶mrna，以阐明它们在干细胞维持、细胞分化和肿瘤发生中的作用。我们应用计算数据挖掘方法在这些Msi1靶向基因的3' UTR中找到调控信号和结构基序。实验和计算结果表明，Msi1的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。基于热力学过程的伪能量最小化模拟算法通常将单个RNA序列的折叠自由能最小化以预测其二级结构。额外使用来自多个序列比对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开发了一种算法RSpredict，它可以预测一组对齐序列的一致二级结构，该算法结合了动态规划原理和协变分数。利用核磁共振、x射线晶体学或其他实验技术来测定大的三维RNA结构一直是一个非常困难的问题。我们的团队与CCR的王云兴团队一起开发了一种结合NMR和SAXS技术的方法，以确定主要由a型螺旋组成的大rna的整体结构。确定螺旋的方向和绕其螺旋轴的旋转以及螺旋的相对全局位置可用于确定结构。SAXS用于确定分子形状的包络，RDC用于确定螺旋的相对取向和旋转相。