Consistent global structures of complex RNA states through multidimensional chemical mapping.
Consistent global structures of complex RNA states through multidimensional chemical mapping.
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作者：
Cheng CY;Chou FC;Kladwang W;Tian S;Cordero P;Das R
在无数生物过程中加速发现非编码RNA（ncRNA）对结构和功能分析提出了重大挑战。尽管在二级结构建模方面取得了进展，但高通量方法通常无法确定ncRNA的三级结构，即使在1 nm分辨率下也无法实现螺旋和功能基序如何在三维空间中定位的可视化。我们报告称，将一种名为MOHCA-seq（具有配对末端测序的多重·OH切割分析）的新方法与突变和映射二级结构推断相结合，指导Rosetta 3D建模，以保持长度高达188个核苷酸的复杂折叠ncRNA的一致1 nm精度，包括盲RNA难题挑战，lariat-capping核酶。这种多维化学映射（MCM）管道解决了意想不到的三级接近的环状二GMP，甘氨酸，腺苷钴胺素核糖开关适配体没有他们的配体和松散的结构，最近发现的人类HoxA 9D内部核糖体进入位点调节子。MCM提供了一种基于测序的途径来揭示ncRNA的3D结构，适用于功能重要但可能异质的状态。DOI：http://dx.doi.org/10.7554/eLife.07600.001我们的遗传物质，以DNA分子的形式，为我们细胞中的许多不同过程提供指令。为了发出这些指令，DNA的特定部分被复制以制造一种称为核糖核酸（RNA）的分子。其中一些RNA分子含有制造蛋白质的指令，但另一些称为非编码RNA的分子则调节细胞中基因的活性。RNA中的遗传信息由四个不同的化学部分编码，称为“核苷酸”。RNA可以以单链核苷酸的形式存在，但核苷酸也可以以特定的组合配对形成双链RNA的片段。因此，非编码RNA的单链可以折叠成复杂的三维形状，其中包含环，扭曲和凸起。非编码RNA的三维结构对于它们在细胞中的作用至关重要，但是它们可以形成的形状的多样性和复杂性使得研究它们在技术上很困难。2008年，研究人员开发了一种名为MOHCA的新方法，可以绘制三维结构中紧密相连的核苷酸的位置。高活性化学物质附着在核苷酸上，这些化学物质可以与附近的其他核苷酸发生反应并破坏它们。通过检测哪些核苷酸被损坏，可以绘制这些核苷酸的位置，并使用计算机算法破译RNA分子的结构。MOHCA是一种很有前途的方法，但最初发现受损核苷酸的方法很繁琐，需要专门的设备。现在，程，达斯等人-包括参与2008年工作的一些研究人员，已经开发出一种改进版的MOHCA，它使用现成的RNA测序技术来找到受损的核苷酸。然后通过Rosetta计算机建模软件中的新算法分析RNA测序数据。Cheng，Das等人使用这种新开发的“MOHCA-seq”和Rosetta揭示了人类非编码RNA和其他几种非编码RNA分子的结构，比以前更详细。MOHCA-seq和Rosetta共同为研究人员提供了一种快速破译非编码RNA三维结构的方法。这种方法可能会加快非编码RNA复杂结构的分析。在未来的工作中，研究这些RNA在细胞中发挥的作用将是有用的，包括它们在癌症、神经变性和其他疾病中的活性。DOI：http://dx.doi.org/10.7554/eLife.07600.002网站
Accelerating discoveries of non-coding RNA (ncRNA) in myriad biological processes pose major challenges to structural and functional analysis. Despite progress in secondary structure modeling, high-throughput methods have generally failed to determine ncRNA tertiary structures, even at the 1-nm resolution that enables visualization of how helices and functional motifs are positioned in three dimensions. We report that integrating a new method called MOHCA-seq (Multiplexed •OH Cleavage Analysis with paired-end sequencing) with mutate-and-map secondary structure inference guides Rosetta 3D modeling to consistent 1-nm accuracy for intricately folded ncRNAs with lengths up to 188 nucleotides, including a blind RNA-puzzle challenge, the lariat-capping ribozyme. This multidimensional chemical mapping (MCM) pipeline resolves unexpected tertiary proximities for cyclic-di-GMP, glycine, and adenosylcobalamin riboswitch aptamers without their ligands and a loose structure for the recently discovered human HoxA9D internal ribosome entry site regulon. MCM offers a sequencing-based route to uncovering ncRNA 3D structure, applicable to functionally important but potentially heterogeneous states. DOI: http://dx.doi.org/10.7554/eLife.07600.001 Our genetic material, in the form of molecules of DNA, provides instructions for many different processes in our cells. To issue these instructions, particular sections of DNA are copied to make a type of molecule called ribonucleic acid (RNA). Some of these RNA molecules contain instructions to make proteins, but others—known as non-coding RNAs—regulate the activity of genes in cells. The genetic information within RNA is encoded by the sequence of four different chemical parts called ‘nucleotides’. RNA can exist as a single strand of nucleotides, but the nucleotides can also pair up in specific combinations to form sections of double-stranded RNA. Therefore, a single strand of non-coding RNA can fold into a complex three-dimensional shape that contains loops, twists, and bulges. The three-dimensional structures of non-coding RNAs are crucial for their roles in cells, but the variety and complexity of shapes that they can form makes it technically difficult to study them. In 2008, researchers developed a new method called MOHCA that can map the positions of nucleotides that are close together in the three-dimensional structure. Highly reactive chemicals are attached to the nucleotides and these can react with, and damage, other nearby nucleotides. By detecting which nucleotides have been damaged, it is possible to map the positions of these nucleotides and decipher the structure of the RNA molecule using computer algorithms. MOHCA is a promising approach, but the initial methods to find the damaged nucleotides were tedious and required specialized equipment. Now, Cheng, Das et al.—including some of the researchers involved in the 2008 work—have developed an improved version of MOHCA that uses readily available RNA sequencing techniques to find the damaged nucleotides. The RNA sequencing data are then analyzed by a new algorithm in the Rosetta computer modeling software. Cheng, Das et al. used this newly developed ‘MOHCA-seq’ and Rosetta to reveal the structures of a human non-coding RNA and several other non-coding RNA molecules to a much higher level of detail than before. Together, MOHCA-seq and Rosetta provide a rapid method for researchers to decipher the three-dimensional structure of non-coding RNAs. This method is likely to speed up the analysis of the complex structures of non-coding RNAs. It will be useful in future efforts to work out what roles these RNAs play in cells, including their activity in cancer, neurodegeneration, and other diseases. DOI: http://dx.doi.org/10.7554/eLife.07600.002