Volume: 15, Issue: 5

Review

RNA Regulations and Functions Decoded by Transcriptome-wide RNA Structure Probing

Meiling Piao, Lei Sun, Qiangfeng Cliff Zhang

RNA folds into intricate structures that are crucial for its functions and regulations. To date, a multitude of approaches for probing structures of the whole transcriptome, i.e., RNA structuromes, have been developed. Applications of these approaches to different cell lines and tissues have generated a rich resource for the study of RNA structure–function relationships at a systems biology level. In this review, we first introduce the designs of these methods and their applications to study different RNA structuromes. We emphasize their technological differences especially their unique advantages and caveats. We then summarize the structural insights in RNA functions and regulations obtained from the studies of RNA structuromes. And finally, we propose potential directions for future improvements and studies.
RNA在在各种各样的生命过程中发挥功能，其细胞内的时间和空间分布都受到严密调控。然而我们对其过程的分子机制知之甚少，因而需要发展新技术来帮助进行深入研究。和蛋白一样，RNA分子也有一级结构即序列、二级结构即碱基配对、三级结构即单个分子空间三维结构、和四级结构即复合体结构等概念。RNA的结构特别是二级结构对于其功能和调控有着十分重要的意义。一方面，RNA结构信息以前在实验上难以得到，传统上主要依赖生物信息学预测。另一方面，从1970年代以来，研究者就发明了各种分子生化技术包括酶切、小分子修饰或者交联来探测RNA结构。在过去的几年时间里，RNA结构分子探测技术与飞速发展的高通量测序结合可以得到大规模甚至全转录组RNA结构（称之为RNA结构组）的信息。在海量信息的基础上，人们发现了许多新的关于RNA加工、翻译等过程的中结构和功能关系，对背后的分子机制比如RNA修饰以及和蛋白质和miRNA结合也有了很多深入了解。然而，这些新方法都还有各自一定的局限性。在很多RNA相关的重大科学问题中，由于方法的局限性难以得到足够的信息进行深入研究。在这篇综述中，我们比较了探测RNA二级结构的不同方法及优劣，探讨了RNA二级结构与RNA功能，基因调控之间的关系，尤其强调了高通量方法在研究RNA结构组及功能中的应用。最后，我们展望了RNA结构与功能研究的未来方向及关注的重大科学问题。

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Review

Engineering Novel Molecular Beacon Constructs to Study Intracellular RNA Dynamics and Localization

Zhao Ma, Xiaotian Wu, Christopher J. Krueger, Antony K. Chen

With numerous advancements in novel biochemical techniques, our knowledge of the role of RNAs in the regulation of cellular physiology and pathology has grown significantly over the past several decades. Nevertheless, detailed information regarding RNA processing, trafficking, and localization in living cells has been lacking due to technical limitations in imaging single RNA transcripts in living cells with high spatial and temporal resolution. In this review, we discuss techniques that have shown great promise for single RNA imaging, followed by highlights in our recent work in the development of molecular beacons (MBs), a class of nanoscale oligonucleotide-probes, for detecting individual RNA transcripts in living cells. With further refinement of MB design and development of more sophisticated fluorescence microscopy techniques, we envision that MB-based approaches could promote new discoveries of RNA functions and activities.
RNA在多种细胞生理过程中的功能是当前生命科学最活跃的前沿研究之一。在过去的数十年中，由于生物化学实验方法的快速发展，RNA在细胞生理学及细胞病理学中的调控作用已经越来越多的被研究和证明。显微成像技术在RNA研究领域的应用对RNA的加工、转运及活细胞定位等相关研究提供便利，然而，技术的局限导致在活细胞中对单个RNA分子转录过程的成像难以获得较高的时间和空间分辨率。在这篇综述中，我们概述了多种单分子RNA成像技术，包括single-molecule Fluorescence in situ hybridization、ms2-GFP系统和基于分子信标的RNA成像系统，其中重点讨论了分子信标（MBs）在单分子RNA成像领域中的应用以及本实验室对分子信标成像系统的改进工作。分子信标是一种纳米级别的寡核苷酸探针，主要用来在活细胞中标记RNA分子，目前已用于检测活细胞中单个RNA的转录过程。随着MB设计的不断改良以及更高分辨率的显微成像技术的出现，我们相信MB技术能帮助研究者对揭示RNA新功能以及疾病检测等相关研究做出贡献。

Page 279-286

Review

Alternative Polyadenylation: Methods, Findings, and Impacts

Wei Chen, Qi Jia, Yifan Song, Haihui Fu, Gang Wei, Ting Ni

Alternative polyadenylation (APA), a phenomenon that RNA molecules with different 3′ ends originate from distinct polyadenylation sites of a single gene, is emerging as a mechanism widely used to regulate gene expression. In the present review, we first summarized various methods prevalently adopted in APA study, mainly focused on the next-generation sequencing (NGS)-based techniques specially designed for APA identification, the related bioinformatics methods, and the strategies for APA study in single cells. Then we summarized the main findings and advances so far based on these methods, including the preferences of alternative polyA (pA) site, the biological processes involved, and the corresponding consequences. We especially categorized the APA changes discovered so far and discussed their potential functions under given conditions, along with the possible underlying molecular mechanisms. With more in-depth studies on extensive samples, more signatures and functions of APA will be revealed, and its diverse roles will gradually heave in sight.
选择性多聚腺苷酸化(APA)，即相同基因转录出具有不同多聚腺苷酸位点(pA位点)的RNA分子，已成为一种普遍的基因表达调控方式。这里我们首先总结进行APA研究用到的各种方法，主要聚焦于专门用于pA位点鉴定的二代测序文库构建方法，相关的生物信息学方法，以及单细胞pA位点鉴定的二代文库构建方法。然后我们总结目前为止APA研究领域的发现和进展，包括pA位点的选择偏好性，这其中涉及到的生物学过程，以及偏好性导致的结果。特别地，我们将已发现的APA变化进行了分类，并且讨论不同类别之中APA变化对基因功能的影响，以及可能的分子机制的影响。随着更深入广泛的研究，更多的APA标志和功能，以及它在生物学过程中的多重角色将会被发现。

Page 287-300

Review

Non-coding RNAs and Their Roles in Stress Response in Plants

Jingjing Wang, Xianwen Meng, Oxana B. Dobrovolskaya, Yuriy L. Orlov, Ming Chen

Eukaryotic genomes encode thousands of non-coding RNAs (ncRNAs), which play crucial roles in transcriptional and post-transcriptional regulation of gene expression. Accumulating evidence indicates that ncRNAs, especially microRNAs (miRNAs) and long ncRNAs (lncRNAs), have emerged as key regulatory molecules in plant stress responses. In this review, we have summarized the current progress on the understanding of plant miRNA and lncRNA identification, characteristics, bioinformatics tools, and resources, and provided examples of mechanisms of miRNA- and lncRNA-mediated plant stress tolerance.
真核生物的基因组可以编码多种非编码RNA（ncRNA）。ncRNA是一群具有低编码蛋白潜在性的RNA。根据他们的长度，可以分为小RNA（smRNA）、中间长度的ncRNA和长非编码RNA（lncRNA）。这些ncRNA在基因的转录和转录后的调控中发挥着重要的作用。越来越多的证据表明非编码RNA，尤其是miRNA和长非编码RNA（lncRNA）已经成为了植物胁迫反应中的关键调控因子。在这篇综述中，我们不仅总结了植物的miRNA和lncRNA的产生过程、序列特征、已有的生物信息学鉴定预测工具、数据库和资源，而且我们探讨了miRNA和lncRNA在植物胁迫应答过程中的可能机制。其中，miRNA可以通过自身的上下调，从而影响靶点的表达量；而lncRNA可以通过五种方式参与免疫应答，包括：作为内源性竞争RNA（ceRNA）、作为其他小RNA前体、与mRNA形成自然反义转录本（NAT）对、介导组蛋白修饰和DNA甲基化。

Page 301-312

Original Research

Latent HIV-1 TAR Regulates 7SK-responsive P-TEFb Target Genes and Targets Cellular Immune Responses in the Absence of Tat

Sebastian Eilebrecht, Bernd-Joachim Benecke, Arndt G. Benecke

The transactivating response element (TAR) structure of the nascent HIV-1 transcript is critically involved in the recruitment of inactive positive transcription elongation factor b (P-TEFb) to the promoter proximal paused RNA polymerase II. The viral transactivator Tat is responsible for subsequent P-TEFb activation in order to start efficient viral transcription elongation. In the absence of the viral transactivator of transcription (Tat), e.g., during latency or in early stages of HIV transcription, TAR mediates an interaction of P-TEFb with its inhibitor hexamethylene bis-acetamide-inducible protein 1 (HEXIM1), keeping P-TEFb in its inactive form. In this study, we address the function of HIV-1 TAR in the absence of Tat by analyzing consequences of HIV-1 TAR overexpression on host cellular gene expression. An RNA chimera consisting of Epstein-Barr virus-expressed RNA 2 (EBER2) and HIV-1 TAR was developed to assure robust overexpression of TAR in HEK293 cells. The overexpression results in differential expression of more than 800 human genes. A significant proportion of these genes is involved in the suppression of cellular immune responses, including a significant set of 7SK-responsive P-TEFb target genes. Our findings identify a novel role for HIV-1 TAR in the absence of Tat, involving the interference with host cellular immune responses by targeting 7SK RNA-mediated gene expression and P-TEFb inactivation.
人类免疫缺陷病毒1（Human Immunodeficiency Virus 1, HIV-1）的新生转录本上存在着反式激活效应元件（transactivating response element, TAR）。这些结构元件在将失活的正性转录延伸因子b（positive transcription elongation factor b, P-TEFb）招募到停顿在启动子近端的RNA聚合酶II的过程中起着关键性作用。随后，病毒的反式激活因子Tat （Transactivator, Tat）激活P-TEFb激活，有效地启动病毒转录延伸。法国巴黎第六大学的Arndt G. Benecke博士等利用EBV病毒表达的RNA2（EBER2）和HIV-1 TAR组合的RNA嵌合体来确保TAR在HEK293细胞中过表达，通过分析TAR过表达对宿主细胞基因表达的影响来探究HIV-1 TAR在缺乏Tat时的功能。通过实验验证，他们识别了HIV-1 TAR在缺乏Tat时的一个新角色——即通过靶向7SK RNA调控的基因表达和P-TEFb失活来干涉宿主细胞免疫反应。
Die TAR-Struktur des entstehenden HIV-1 Transkripts ist wesentlich daran beteiligt, inaktiven P-TEFb zu der Promoter-nah pausierenden RNA Polymerase II zu rekrutieren. Der virale Transaktivator Tat ist anschließend dafür verantwortlich, dass P-TEFb aktiviert wird und die effiziente virale Transkription initiieren kann. In der Abwesenheit von Tat, wie es zum Beispiel während der viralen Latenz oder in frühen Phasen der HIV Transkription der Fall ist, vermittelt TAR die Interaktion von P-TEFb mit seinem Inhibitor HEXIM1, was dazu führt, dass P-TEFb inaktiv bleibt. In der vorliegenden Studie untersuchen wir die Funktion von HIV-1 TAR in der Abwesenheit von Tat anhand der Analyse von zellulären Genexpressionsänderungen, welche durch eine HIV-1 TAR Überexpression hervorgerufen werden. Dazu haben wir eine RNA-Chimäre hergestellt, welche aus der EBER2 RNA des Epstein Barr Virus und der HIV-1 TAR RNA besteht und somit robust exprimiert wird. In HEK293 Zellen führt eine solche TAR Überexpression zu einer Änderung der Expression von über 800 humanen Genen, von denen ein erheblicher Anteil an der Unterdrückung der zellulären Immunantwort beteiligt ist, darunter auch eine Reihe von 7SK-regulierten P-TEFb Zielgenen. Unsere Ergebnisse zeigen eine neue Rolle von HIV-1 TAR in der Abwesenheit von Tat, in der TAR über die Veränderung der 7SK-vermittelten Genexpression und die Inaktivierung von P-TEFb in die zelluläre Immunantwort des Wirts eingreift.

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Letter

Ribosome Profiling Reveals Genome-wide Cellular Translational Regulation upon Heat Stress in Escherichia coli

Yanqing Zhang, Zhengtao Xiao, Qin Zou, Jianhuo Fang, Qifan Wang, Xuerui Yang, Ning Gao

Heat shock response is a classical stress-induced regulatory system in bacteria, characterized by extensive transcriptional reprogramming. To compare the impact of heat stress on the transcriptome and translatome in Escherichia coli, we conducted ribosome profiling in parallel with RNA-Seq to investigate the alterations in transcription and translation efficiency when E. coli cells were exposed to a mild heat stress (from 30 °C to 45 °C). While general changes in ribosome footprints correlate with the changes of mRNA transcripts upon heat stress, a number of genes show differential changes at the transcription and translation levels. Translation efficiency of a few genes that are related to environment stimulus response is up-regulated, and in contrast, some genes functioning in mRNA translation and amino acid biosynthesis are down-regulated at the translation level in response to heat stress. Moreover, our ribosome occupancy data suggest that in general ribosomes accumulate remarkably in the starting regions of ORFs upon heat stress. This study provides additional insights into bacterial gene expression in response to heat stress, and suggests the presence of stress-induced but yet-to-be characterized cellular regulatory mechanisms of gene expression at translation level.
热激反应是细菌中经典的应激调控系统，主要表现为基因转录在热激状态时有大尺度的重编程过程。针对热激反应转录水平的调控，基于DNA芯片和RNA-seq技术已经广泛应用并获得了大量的进展。与此相比的是，细胞在热激反应时mRNA翻译水平的组学研究相对较少，翻译水平的调控机理尚不清楚。核糖体翻译谱分析技术（Ribosome Profiling）可以同时定量分析细胞内的处于翻译进程中的所有mRNA，可以较为准确地评估细胞全基因组的基因翻译效率以及单个基因的翻译调控事件。 为了对比大肠杆菌在热激状态下转录组和翻译组的调控变化，我们利用ribosome profiling和RNA-seq技术研究了大肠杆菌在正常和热激条件下的基因转录和翻译效率。研究发现，虽然多数基因在热激前后核糖体翻译效率变化和转录水平变化基本一致，但仍有部分基因在翻译水平和转录水平变化倍数差异较大或变化趋势相反。具体来说，与环境变化应激反应有关的一些基因在翻译水平上调，相反地，蛋白质生成及氨基酸合成通路的一些基因在热激之后翻译效率明显下调。而且，结果表明，热激之后核糖体普遍会在mRNA翻译起始区域附近积累。此研究增进了我们对细菌在热激反应中的基因调控的认识，为进一步研究特异的翻译水平的基因表达调控通路提供了线索。

Page 324-330

Perspective

Steering Against Wind: A New Network of NamiRNAs and Enhancers

Ying Liang, Qingping Zou, Wenqiang Yu

MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs with regulatory functions. Traditionally, miRNAs are thought to play a negative regulatory role in the cytoplasm by binding to the 3′UTR of target genes to degrade mRNA or inhibit translation. However, it remains a challenge to interpret the potential function of many miRNAs located in the nucleus. Recently, we reported a new type of miRNAs present in the nucleus, which can activate gene expression by binding to the enhancer, and named them nuclear activating miRNAs (NamiRNAs). The discovery of NamiRNAs showcases a complementary regulatory mechanism of miRNA, demonstrating their differential roles in the nucleus and cytoplasm. Here, we reviewed miRNAs in nucleus to better understand the function of NamiRNAs in their interactions with the enhancers. Accordingly, we propose a NamiRNA–enhancer–target gene activation network model to better understand the crosstalk between NamiRNAs and enhancers in regulating gene transcription. Moreover, we hypothesize that NamiRNAs may be involved in cell identity or cell fate determination during development, although further study is needed to elucidate the underlying mechanisms in detail.
microRNA（以下简称miRNA）是长度约21-23个核苷酸的内源性非编码单链RNA，广泛存在于动物、植物、微生物等生物体中，参与细胞与组织的生长、发育、分化以及疾病的发生发展中。因此研究miRNA的调控机制具有重要的理论意义和应用价值。从1993年第一个miRNA的发现，至今已有30多个年头，传统意义上认为miRNA在细胞浆中通过结合靶基因3’UTR抑制翻译或降解mRNA进而发挥负向调控作用，然而不足以解释所有由miRNA调控介导的生理现象，miRNA的正向激活功能也逐渐被发现及报道。另一方面，随着miRNA研究的不断深入和深度测序技术的发展，越来越多的证据表明，miRNA不仅定位于细胞浆，而且也存在于细胞核。我们相信这些定位于细胞核内的miRNA在基因的表达调控过程中发挥着重要作用，而且这些miRNA所发挥的功能应该不同于传统的miRNA发挥的负向调控作用。

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