环状RNA（circular RNA,circRNA）是一种在转录过程中产生的单链环形RNA。与常见的线性剪接（linear splicing）方式有所不同,环状RNA通过反向剪接（back splicing）形成,即在加工过程中,前体RNA外显子或内含子的5’和3’端相互靠近,经剪接体加工后首尾相接,形成共价闭合的环状分子（Kristensen et al. 2019）。环状RNA普遍存在于众多物种中,在人类、小鼠、线虫以及真菌中,已经鉴定出数以千计的环状RNA。目前关于环状RNA表达谱的研究表明,环状RNA的表达不仅呈现出明显的物种特异性,而且在不同组织器官以及不同发育时期也具有一定的时空特异性（Chen 2020）。此外,一部分环状RNA在进化过程中表现出一定的保守性,其同源序列在人类和小鼠中皆有表达（Memczak et al. 2013）。这种生物普遍性和多样性提示环状RNA很可能具有丰富的分子生物学功能（Salzman et al. 2012）。
环状RNA分子呈闭合环状结构,不易降解,可以稳定地存在于胞浆（Huang et al. 2018）。同时,环状RNA功能十分丰富,可以充当微小RNA（microRNA）分子海绵、蛋白质分子海绵、蛋白质分子脚手架和翻译模板等,因而在基因转录、转录后修饰、蛋白质翻译以及翻译后修饰等多个层次均可发挥调控作用（Chen 2020;Shao et al. 2021）。现有研究表明,环状RNA不仅在诸如细胞分化、组织发育等正常生理调控作用中具有重要角色,而且在如细胞增殖异常、细胞代谢异常等多种病理过程中也发挥调控作用（Kristensen et al. 2019）。因此,深度解析环状RNA的功能,对于揭示转录组相互作用、研究生物体的复杂调控过程以及生物遗传进化机制等具有重要意义。
1 真菌环状RNA的研究现状及难点
虽然环状RNA逐渐成为RNA领域中的研究热点,但其在真菌中的研究却仍然有限。目前,在真菌中仅仅报道了数十至数千不等的环状RNA（表1）,而且这些研究主要局限在一些模式菌种,如蜜蜂球囊菌Ascosphaera apis（Guo et al. 2018a）、新生隐球菌Cryptococcus neoformans（Huo et al. 2018）、灵芝Ganoderma lucidum（Shao et al. 2019）、稻瘟菌Magnaporthe oryzae（Yuan et al. 2018）和裂殖酵母Schizosaccharomyces pombe （Wang et al. 2014）等。此外,虽然灵芝以及皮炎外瓶霉Exophiala dermatitidis等菌株在不同生长条件下的环状RNA表达谱差异提示环状RNA可能参与了真菌生长、发育、分化等过程（Blasi et al. 2015;Poyntner et al. 2016;Shao et al. 2019）,但是与高等真核生物的环状RNA研究相比,目前真菌中环状RNA的研究仍然相对简单,主要停留于环状RNA的鉴定以及表达差异的定性比较,缺乏揭示真菌环状RNA的分子功能及其调控机制的研究。
表1 不同真菌环状RNA研究比较
Table 1
| 物种 Species | 富集方法 Enrichment method | 软件 Software | 环状RNA种类 Type of circular RNA | 参考文献 Reference |
|---|---|---|---|---|
| Schizosaccharomyces pombe | Total RNA | In house scripts | 42 | Wang et al. 2014 |
| Exophiala dermatitidis | polyA(+) | In house scripts | 87-677 | Blasi et al. 2015 |
| Exophiala dermatitidis | polyA(+) | In house scripts | 62-126 | Poyntner et al. 2016 |
| Ascosphaera apis | Ribo-Zero rRNA removal kit | CIRI | 551 | Guo et al. 2018a |
| Magnaporthe oryzae | Ribo-Zero gold kit/RNase R | Find_circ | 8 848 | Yuan et al. 2018 |
| Cryptococcus neoformans | RibominusTM transcriptome isolation kit/RNase R | CIRI | 73 | Huo et al. 2018 |
| Nosema ceranae | Ribo-Zero rRNA removal kit | CIRI | 204 | Guo et al. 2018b |
| Ganoderma lucidum | polyA(-) and polyA(-)/RNase R | CIRCexplorer2 circRNA_finder CIRI2 | 250-2 193 | Shao et al. 2019 |
究其原因,缺乏成熟的真菌环状RNA富集分析流程是限制真菌环状RNA研究进展的重要原因。一方面,真菌具有基因组较小、外显子数量较少、压缩度较高等特点,因而其产生的环状RNA数目可能远少于人类等高等真核生物。此外,真菌细胞壁的存在增加了RNA提取的技术难度。这些因素都导致适用于人类等高等真核生物的环状RNA实验处理流程在真菌中并不能发挥理想的富集效果。而真菌环状RNA的富集水平较低则在一定程度上影响了后续流程中环状RNA鉴定以及差异分析的准确性。另一方面,目前环状RNA鉴定的生物信息学软件大部分需要依赖物种自身的基因组序列及其注释信息,而大部分真菌缺少高质量的基因组序列及其注释信息,特别是对于一些非模式真菌,其基因组序列和注释信息尚不完整,这也是限制诸多非模式真菌环状RNA研究的关键因素。
除上述因素外,真菌环状RNA的形成方式与哺乳动物也存在不同之处,在一定程度上也增加了真菌环状RNA的研究难度。由于内含子较长,人类或小鼠等哺乳动物在RNA加工过程中,剪接体主要采用“跨外显子”的组装方式来发挥剪接作用。因此,大部分人类环状RNA的形成依赖存在于内含子侧翼的反向重复互补序列（Kristensen et al. 2019）。该序列在前体RNA处理时诱导局部双链互补茎状结构产生,茎状结构被剪接体进一步剪接后即产生环状RNA。但真菌基因组具有压缩程度高、基因密集、内含子短而少的特点,剪接体则倾向采用“跨内含子”组装方式加工RNA。因此,基因结构的差异导致两者剪接体作用机制产生差别（Plaschka et al. 2018）。在酵母中,其主要是通过含有外显子的套索前体诱导产生环状RNA （Barrett et al. 2015）。套索前体模型可能通过减少环状RNA与线性转录本竞争剪接位点,从而促进环化。但这种套索诱导的环化方式是否普遍存在于其他真菌中则需要更多的研究来证明。
2 真菌环状RNA的筛选与鉴定
环状RNA的筛选与鉴定步骤大致分为实验富集、高通量测序、生物信息学分析以及实验验证4个环节。
2.1 环状RNA的实验富集方法
大部分环状RNA的表达量相对较低,除极少数环状RNA具有较高的表达水平外（You et al. 2015;Zheng et al. 2016）,大多数环状RNA的丰度不超过相应线性转录本的10%（Salzman et al. 2013;Guo et al. 2014）。因此,环状RNA的富集过程是研究环状RNA表达及功能不可或缺的环节。由于大多数环状RNA没有特定的多聚腺苷酸尾修饰（Memczak et al. 2013）,且长度一般在200-1 000bp之间,因此无法利用ploy(T)磁珠纯化或者小片段切胶回收的方式进行富集。由于大多数环状RNA缺乏自由末端而可以抵抗核酸酶RNase R降解,因此可以通过RNase R降解线性RNA,从而达到富集环状RNA的效果。此外,在去除核糖体RNA建库的基础上,利用ploy(T)磁珠去除多聚腺苷酸尾线性RNA (ploy(A)-),也可以提高环状RNA的比例。值得注意的是,最近有研究指出,环状RNA中存在部分对RNase R敏感或者具有ploy(A)尾的亚群（Szabo & Salzman 2016）,因而上述富集方法可能会降低这些环状RNA的检出率,带来后续的分析偏差。
2.2 环状RNA的高通量测序
目前常用的系统性鉴定环状RNA的方法是将富集的RNA样本建库并进行高通量测序,从而通过识别测序数据中环状RNA特有的反向剪接区域来检测环状RNA。除常用的高通量测序技术外,还可以针对环状RNA反向剪接区域设计探针,从而利用微阵列（microarray）芯片技术检测环状RNA。研究策略可根据不同研究目的进行选择,并对接不同的下游分析流程。
通过第二代高通量测序平台产生单端或双端RNA-seq数据,并借助相应的生物信息学软件识别基因组序列中反向剪接区域,是目前鉴定环状RNA最为常用的方法。然而,相比于具有较高丰度的线性剪接区域,反向剪接区域的丰度则相对较低,在一定程度上限制了环状RNA的研究进展。因此,需要通过实验方法富集环状RNA,从而提升环状RNA的识别效率。此外,适当增加测序深度也是提高环状RNA检出率的主要方法。需要注意的是,测序平台和参数选择同样可能对环状RNA的识别产生影响（Liu et al. 2018;Xin et al. 2021;Zhang et al. 2021）。
除利用RNA-seq数据筛选鉴定环状RNA进行外,针对相应环状RNA的反向剪接部位设计微阵列芯片探针,同样可用于分析组织或者细胞中环状RNA的表达情况（Yang et al. 2020）。不同于RNA-seq,微阵列可以针对特定的环状RNA进行探针设计,从而靶向性的分析某类环状RNA的表达特点（Li et al. 2018）。由于微阵列技术成本相对较低,因而这种方法使得环状RNA成为一类具有较大应用潜力的临床诊断的分子标记物。但环状RNA微阵列受限于已知环状RNA种类,不适用于寻找和发现新的环状RNA。
2.3 环状RNA的生物信息学分析
CIRI2（Gao et al. 2018）、CIRCexplorer3 （Ma et al. 2019）、circRNA_finder （Westholm et al. 2014）、find_circ （Memczak et al. 2013）和KNIFE（Szabo et al. 2015）等工具是基于二代RNA-seq数据中反向剪接读段所开发的环状RNA预测软件。这些软件主要是利用物种注释文件信息建立反向剪接索引库,并识别测序数据中与之匹配的反向剪接区域,从而预测由反向剪接所形成的环状RNA。此外,常规GT-AG剪接位点的识别也有助于精确定位环状RNA的反向剪接部位。但某些环状RNA的剪接位点并非常规的GT-AG,例如剪接自内含子的环状RNA。因而,仅靠识别常规剪接位点会遗失特殊种类的环状RNA（Zhang et al. 2021）。同时,受限于二代RNA-seq数据读长限制,无法准确提供环状RNA的全长序列,因而限制了环状RNA的空间结构和可变剪接的相关研究。
对环状RNA的定量分析有利于功能性环状RNA的鉴定。然而,目前环状RNA的识别主要集中于反向剪接区域,对于非剪接区域的读段则难以排除来自其宿主基因的干扰。因此,包括FPKM和TPM在内的转录组定量指标通常不适用于环状RNA差异表达分析。为了降低环状RNA分析过程中宿主基因表达带来的影响,近年来的研究利用反向剪接读段与正向剪接读段的比值,即环状RNA与宿主基因表达量之比,来校正环状RNA的表达量（Zhang et al. 2020;Liu et al. 2021）。这种相对含量定量方式可以有效地降低环状RNA差异分析的假阳性率,从而提供更可靠的环状RNA候选集。但是由于环状RNA建库测序过程中通常会利用RNase R降解线性RNA,这也会导致这种相对定量的方法存在偏倚。CIRI-quant（Zhang et al. 2020）软件以及一些其他的分析流程（Vo et al. 2019）提供了校正RNase R处理偏倚的统计模型,可以通过对比RNase R处理组与RNase R非处理组,获得更准确的circRNA表达量。
2.4 环状RNA的分子生物学验证与定量
虽然基于RNA-seq的生物信息学分析可以基本满足不同样本间环状RNA差异分析的筛选需求,但其结果常常受到富集偏差、测序偏差、算法偏差等多方面因素的影响。因此,通常还需要从分子水平上对所预测的环状RNA进行验证,以评估生物信息学软件的预测效果。Northern印迹法是验证circRNA的金标准（Li et al. 2015b;Kristensen et al. 2019）,但其较高的环状RNA投入量和具有标记探针的放射性限制了其应用范围。目前,针对反向剪接区域设计特异性的反向引物是验证环状RNA最为常用的验证手段之一,将RNase R处理与反向引物扩增相结合即可有效地验证环状RNA存在（Memczak et al. 2013）。此外,利用反向引物对环状RNA进行滚环复制（rolling circle amplification,RCA）,可以得到环状RNA全长序列（You et al. 2015）,不仅可验证反向剪接位点,也为进一步研究环状RNA的环化机制或者与其他分子的互作结合位点提供了基础。
在具体环状RNA分子的研究中,实验定量有助于进一步确定目标环状RNA的表达差异情况,为后续通过遗传操作等分子生物学手段研究其功能提供了实验基础。在分子水平上对环状RNA的定量可以采用基于反向引物的RT-qPCR方法。然而,由于环状RNA的PCR扩增过程中存在滚环复制和模板转换的现象,导致这种定量方法可能存在一定程度的偏倚（Szabo & Salzman 2016）。利用放射性反向探针则可以克服这种偏差（Hansen et al. 2013）。此外,使用SplintQuant （Conn & Conn 2019）等优化定量实验流程也可实现对环状RNA的精确定量。
3 真菌环状RNA的研究展望
真菌中关于环状RNA的研究尚处于起步阶段,目前还没有功能性环状RNA在真菌中被报道。现有研究中所发现的真菌环状RNA数目远少于其他物种：一方面,真菌基因组内含子少,基因排列紧密可能导致真菌产生的环状RNA总数较少;另一方面,缺少成熟的实验流程和分析流程,也限制了真菌环状RNA的研究。因此,建立成熟的实验流程以及高效、灵敏、准确的环状RNA分析方法,是进一步解析真菌环状RNA的重要环节。
现有的真菌环状RNA的研究显示,核糖体去除的RNA-seq建库方法可以明显提高环状RNA的检出数目（Guo et al. 2018a,2018b）。同时,RNase R以及ploy(A)-处理对于提高环状RNA的检出率也具有一定的附加效果（Huo et al. 2018;Yuan et al. 2018;Shao et al. 2019）。但是由于缺少相应的对照实验,尚不能评估最佳的富集流程。真菌环状RNA研究主要基于二代测序数据和主流环状RNA预测软件,如CIRI2等。然而,由于物种差异、前期实验处理不同以及筛选阈值的选择不同,难以就目前结果对真菌环状RNA进行准确的数量和质量评估。随着该领域的不断发展和数据的不断丰富,对于真菌环状RNA的评估也会实现质的飞跃。
目前,环状RNA的调节功能已在多个物种中得到证实,其充当RNA分子海绵、结合蛋白质、翻译微肽等功能在众多生理病理的发生发展过程中具有重要作用。以往研究证明,lncRNA、miRNA等非编码RNA参与了真菌的细胞分裂、营养感知、有性生殖等过程（Chacko et al. 2015;Zeng et al. 2019;Liu et al. 2020）,考虑到相当数量的环状RNA具有miRNA结合位点（Memczak et al. 2013;Piwecka et al. 2017）或与蛋白的结合能力（Ashwal-Fluss et al. 2014;Abdelmohsen et al. 2017）,环状RNA在真菌转录组的复杂调控网络中可能同样发挥重要作用。此外,一些病原真菌与宿主相互作用时会通过分泌miRNA实现免疫逃逸或毒力调控（Schorey et al. 2015;Cai et al. 2018）。而环状RNA的共价闭合结构使其较线性RNA更为稳定,因而可以在胞外稳定存在,或者在外泌体中明显富集（Li et al. 2015a）。但是,目前尚未有研究涉及真菌中分泌环状RNA领域。
环状RNA作为一类具有强大调节潜力的非编码RNA,在真核生物界具有广泛的调控作用。对真菌环状RNA的功能研究有助于深入解析某些复杂生命过程,例如有性生殖和毒力增强等。这些不仅可以为抗真菌药物的研究提供新的靶点,而且也为真菌基因表达调控机制的研究提供数据基础。
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DOI:10.1038/nsmb.2959 URL [本文引用: 1]
Transcriptomic analysis of extracellular RNA governed by the endocytic adaptor protein Cin1 of Cryptococcus deneoformans
DOI:10.3389/fcimb.2020.00256 URL [本文引用: 1]
Biclustering of transcriptome sequencing data reveals human tissue-specific circular RNAs
DOI:10.1186/s12864-017-4335-9 URL [本文引用: 1]
DEBKS: a tool to detect differentially expressed circular RNA
CIRCexplorer3: a clear pipeline for direct comparison of circular and linear RNA expression
Circular RNAs are a large class of animal RNAs with regulatory potency
DOI:10.1038/nature11928 URL [本文引用: 5]
Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function
DOI:10.1126/science.aam8526 URL [本文引用: 1]
Prespliceosome structure provides insights into spliceosome assembly and regulation
DOI:10.1038/s41586-018-0323-8 URL [本文引用: 1]
The transcriptome of Exophiala dermatitidis during ex-vivo skin model infection
Cell-type specific features of circular RNA expression
DOI:10.1371/journal.pgen.1003777 URL [本文引用: 1]
Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types
DOI:10.1371/journal.pone.0030733 URL [本文引用: 1]
Exosomes and other extracellular vesicles in host-pathogen interactions
DOI:10.15252/embr.201439363 URL [本文引用: 1]
Identification and characterization of circular RNAs in Ganoderma lucidum
DOI:10.1038/s41598-019-52932-w URL [本文引用: 4]
Circular RNA: an important player with multiple facets to regulate its parental gene expression. Molecular Therapy-
Statistically based splicing detection reveals neural enrichment and tissue-specific induction of circular RNA during human fetal development
DOI:10.1186/s13059-015-0690-5 URL [本文引用: 1]
Detecting circular RNAs: bioinformatic and experimental challenges
DOI:10.1038/nrg.2016.114 URL [本文引用: 2]
The landscape of circular RNA in cancer
DOI:10.1016/j.cell.2018.12.021 URL [本文引用: 1]
Circular RNA is expressed across the eukaryotic tree of life
DOI:10.1371/journal.pone.0090859 URL [本文引用: 2]
Genome-wide analysis of drosophila circular RNAs reveals their structural and sequence properties and age-dependent neural accumulation
DOI:S2211-1247(14)00931-0
PMID:25544350
[本文引用: 1]
Circularization was recently recognized to broadly expand transcriptome complexity. Here, we exploit massive Drosophila total RNA-sequencing data, >5 billion paired-end reads from >100 libraries covering diverse developmental stages, tissues, and cultured cells, to rigorously annotate >2,500 fruit fly circular RNAs. These mostly derive from back-splicing of protein-coding genes and lack poly(A) tails, and the circularization of hundreds of genes is conserved across multiple Drosophila species. We elucidate structural and sequence properties of Drosophila circular RNAs, which exhibit commonalities and distinctions from mammalian circles. Notably, Drosophila circular RNAs harbor >1,000 well-conserved canonical miRNA seed matches, especially within coding regions, and coding conserved miRNA sites reside preferentially within circularized exons. Finally, we analyze the developmental and tissue specificity of circular RNAs and note their preferred derivation from neural genes and enhanced accumulation in neural tissues. Interestingly, circular isoforms increase substantially relative to linear isoforms during CNS aging and constitute an aging biomarker. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
isoCirc catalogs full-length circular RNA isoforms in human transcriptomes
DOI:10.1038/s41467-020-20459-8 URL [本文引用: 1]
Extracellular vesicle-mediated delivery of circular RNA scmh1 promotes functional recovery in rodent and nonhuman primate ischemic stroke models
DOI:10.1161/CIRCULATIONAHA.120.045765
PMID:32441115
[本文引用: 1]
Stroke is a leading cause of adult disability that can severely compromise the quality of life of patients, yet no effective medication currently exists to accelerate rehabilitation. A variety of circular RNA (circRNA) molecules are known to function in ischemic brain injury. Lentivirus-based expression systems have been widely used in basic studies of circRNAs, but safety issues with such delivery systems have limited exploration of the potential therapeutic roles for circRNAs.Circular RNA SCMH1 (circSCMH1) was screened from the plasma of patients with acute ischemic stroke by using circRNA microarrays. Engineered rabies virus glycoprotein-circSCMH1-extracellular vesicles were generated to selectively deliver circSCMH1 to the brain. Nissl staining was used to examine infarct size. Behavioral tasks were performed to evaluate motor functions in both rodent and nonhuman primate ischemic stroke models. Golgi staining and immunostaining were used to examine neuroplasticity and glial activation. Proteomic assays and RNA-sequencing data combined with transcriptional profiling were used to identify downstream targets of circSCMH1.CircSCMH1 levels were significantly decreased in the plasma of patients with acute ischemic stroke, offering significant power in predicting stroke outcomes. The decreased levels of circSCMH1 were further confirmed in the plasma and peri-infarct cortex of photothrombotic stroke mice. Beyond demonstrating proof-of-concept for an RNA drug delivery technology, we observed that circSCMH1 treatment improved functional recovery after stroke in both mice and monkeys, and we discovered that circSCMH1 enhanced the neuronal plasticity and inhibited glial activation and peripheral immune cell infiltration. CircSCMH1 binds mechanistically to the transcription factor MeCP2 (methyl-CpG binding protein 2), thereby releasing repression of MeCP2 target gene transcription.Rabies virus glycoprotein-circSCMH1-extracellular vesicles afford protection by promoting functional recovery in the rodent and the nonhuman primate ischemic stroke models. Our study presents a potentially widely applicable nucleotide drug delivery technology and demonstrates the basic mechanism of how circRNAs can be therapeutically exploited to improve poststroke outcomes.
Neural circular RNAs are derived from synaptic genes and regulated by development and plasticity
You, Xintian; Babic, Ana; Quedenau, Claudia; Wang, Xi; Hou, Jingyi; Liu, Hongyu; Sun, Wei; Chen, Tao; Chen, Wei Max Delbruck Ctr Mol Med, Berlin Inst Med Syst Biol, Berlin, Germany. Vlatkovic, Irena; Will, Tristan; Epstein, Irina; Tushev, Georgi; Akbalik, Gueney; Wang, Mantian; Glock, Caspar; Sambandan, Sivakumar; Schuman, Erin M. Max Planck Inst Brain Res, Dept Synapt Plast, Frankfurt, Germany.
Genome-wide identification and characterization of circular RNAs in the rice blast fungus Magnaporthe oryzae
DOI:10.1038/s41598-018-25242-w URL [本文引用: 3]
Cross-Kingdom small RNAs among animals, plants and microbes
DOI:10.3390/cells8040371 URL [本文引用: 1]
Accurate quantification of circular RNAs identifies extensive circular isoform switching events
DOI:10.1038/s41467-019-13840-9 URL [本文引用: 2]
Comprehensive profiling of circular RNAs with nanopore sequencing and CIRI-long
DOI:10.1038/s41587-021-00842-6 URL [本文引用: 2]
Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs
DOI:10.1038/ncomms11215 URL [本文引用: 1]
