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浙江大学学报(医学版)  2022, Vol. 51 Issue (3): 362-372    DOI: 10.3724/zdxbyxb-2022-0017
综述     
活细胞RNA成像技术及其在生物医学中应用研究进展
孙萍萍1,2,邹炜1,2,*()
1. 浙江大学医学院附属第四医院,浙江 义乌 322000
2. 浙江大学医学院转化医学研究院,浙江 杭州 310058
Research progress of live-cell RNA imaging techniques
SUN Pingping1,2,ZOU Wei1,2,*()
1. The Fourth Affiliated Hospital, Zhejiang University School of Medicine, Yiwu 322000, Zhejiang Province, China;
2. Institute of Translational Medicine, Zhejiang University, Hangzhou 310058, China
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摘要:

RNA可与各种核酸及蛋白质相互作用,在多种生理和病理过程中发挥重要的调控作用,其生物学过程高度动态。追踪活细胞中的RNA动态对理解基因表达的时空调控以及RNA的调控功能至关重要。基于荧光蛋白的RNA标记系统包括MS2/MCP系统、PP7/PCP系统、boxB/λN22和CRISPR-Cas系统等,其中MS2/MCP系统目前应用最为广泛,其具有结合稳定、信噪比高等优点,局限性是RNA成像的实现需要对靶RNA进行基因编辑,这可能导致靶RNA特性的潜在改变。近期开发的CRISPR-dCas13系统不需要对RNA进行改造,但其局限性在于CRISPR RNA效率的不确定性,且信噪比较低。基于荧光染料的RNA标记系统包括分子信标和荧光基团-适体对,其中分子信标具有高特异性和较高信噪比,而荧光基团–适体对Pepper系统和Mango系统在RNA成像的信噪比上更具有优势,但也需要对靶RNA进行基因编辑。活细胞RNA成像技术可应用于观察并研究RNA转录、剪接、转运、翻译(特指信使RNA)和亚细胞定位,有助于研究细胞分化等生物学过程以及细胞适应外界环境时的转录调控机制,有利于理解RNA行为异常导致的各种疾病的致病机制以及寻找潜在的治疗靶点。本文综述了活细胞RNA成像技术的发展和运用,并比较了各种方法的优势和不足。

关键词: 活细胞RNA成像基因表达转录调控信使RNA非编码RNA;综述    
Abstract:

RNA molecules play diverse roles in many physiological and pathological processes as they interact with various nucleic acids and proteins. The various biological processes of RNA are highly dynamic. Tracking RNA dynamics in living cells is crucial for a better understanding of the spatiotemporal control of gene expression and the regulatory roles of RNA. Genetically encoded RNA-tagging systems include MS2/MCP, PP7/PCP, boxB/λN22 and CRISPR-Cas. The MS2/MCP system is the most widely applied, and it has the advantages of stable binding and high signal-to-noise ratio, while the realization of RNA imaging requires gene editing of the target RNA, which may change the characteristics of the target RNA. Recently developed CRISPR-dCas13 system does not require RNA modification, but the uncertainty in CRISPR RNA (crRNA) efficiency and low signal-to-noise ratio are its limitations. Fluorescent dye-based RNA-tagging systems include molecular beacons and fluorophore-binding aptamers. The molecular beacons have high specificity and high signal-to-noise ratio; Mango and Peppers outperform the other RNA-tagging system in signal-to-noise, but they also need gene editing. Live-cell RNA imaging allows us to visualize critical steps of RNA activities, including transcription, splicing, transport, translation (for message RNA only) and subcellular localization. It will contribute to studying biological processes such as cell differentiation and the transcriptional regulation mechanism when cells adapt to the external environment, and it improves our understanding of the pathogenic mechanism of various diseases caused by abnormal RNA behavior and helps to find potential therapeutic targets. This review provides an overview of current progress of live-cell RNA imaging techniques and highlights their major strengths and limitations.

Key words: Live-cell RNA imaging    Gene expression    Transcriptional regulation    Message RNA    Non-coding RNA; Review
收稿日期: 2022-01-23 出版日期: 2022-09-21
CLC:  R394  
基金资助: 国家自然科学基金(31970919)
通讯作者: 邹炜     E-mail: zouwei@zju.edu.cn
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孙萍萍,邹炜. 活细胞RNA成像技术及其在生物医学中应用研究进展[J]. 浙江大学学报(医学版), 2022, 51(3): 362-372.

SUN Pingping,ZOU Wei. Research progress of live-cell RNA imaging techniques. J Zhejiang Univ (Med Sci), 2022, 51(3): 362-372.

链接本文:

https://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2022-0017        https://www.zjujournals.com/med/CN/Y2022/V51/I3/362

图 1  MS2/MCP系统成像技术原理示意图融合表达荧光蛋白的MCP二聚体与MS2茎环结合实现RNA成像. MCP:MS2衣壳蛋白.
图 2  PP7/PCP系统成像技术原理示意图融合表达荧光蛋白的PCP二聚体与PP7茎环结合实现RNA成像. PCP:PP7衣壳蛋白.
图 3  boxB/λN22系统成像技术原理示意图融合表达荧光蛋白的λN22单体与boxB茎环结合实现RNA成像.
图 4  CRISPR/dCas13系统成像技术原理示意图在crRNA介导下,融合表达荧光蛋白的dCas13蛋白靶向RNA,实现RNA成像. CRISPR: 成簇的、规律间隔的短回文重复序列; crRNA:CRISPR RNA;dCas:失活的Cas.
图 5  分子信标成像技术原理示意图分子信标探针的两端分别是荧光基团和猝灭基团,一旦与靶RNA结合,两端分开,荧光基团发光.
图 6  RNA适体成像原理示意图单独的染料和RNA适体都不发光,而两者结合会迸发出强烈的荧光.

技术名称

优点

局限

文献

基于荧光蛋白

?

MS2/MCP系统

MCP与MS2结合稳定,标记RNA效率较高,目前运用最为广泛

游离的荧光蛋白产生高背景荧光,需要对靶RNA进行改造,标签的插入可能导致靶RNA的特性改变

[810-12]

PP7/PCP系统

PCP与PP7结合效率高,信噪比较高

[13]

boxB/λN22系统

λN22蛋白小,4×boxB标签已能实现RNA成像,其尺寸小、对靶RNA潜在影响小

[14-15]

CRISPR-dCas13系统

无需对靶基因进行编辑

crRNA效率不确定,难以实现单分子RNA成像,信噪比低

[16]

基于荧光染料

?

分子信标

具有高特异性和较高信噪比

需要显微注射或引入细胞

[17]

荧光基团–适体对

背景荧光低,信噪比高

需要对靶基因进行编辑,适体结构不稳定,需添加染料

[8101518-20]

表 1  活细胞RNA成像技术
图 7  神经元活细胞RNA转录、转运、定位以及翻译过程成像示意图利用活细胞成像技术,在神经元的细胞核中实现转录过程的可视化;也能实现树突和轴突中mRNA的转运、正确定位以及翻译过程的可视化. mRNA:信使RNA.

RNA活动过程

应用系统

成像RNA

应用物种

研究发现

文献

转录

MS2/MCP

act-5csaA新生RNA

盘基网柄菌细胞

与发育基因不同,管家基因通过调节转录爆发强度来响应发育需求

[12]

MS2/MCP

sygl-1新生RNA

秀丽隐杆线虫

Notch信号通过调节转录爆发持续时间来调节下游基因的表达

[59]

MS2/MCP和PP7/PCP

GAPC2HSP70新生RNA

烟草、拟南芥

植物组织通过提高可转录细胞的比率来上调整个组织的表达水平

[60]

mRNA转运和定位

PP7/PCP

erm-1let-413ajm-1 mRNA

秀丽隐杆线虫

mRNA定位在上皮极化和成熟过程不对称分布

[35]

boxB/λN22

ASH1IST2 mRNA

酵母

不同的mRNA被共同组装成RNP 颗粒,定向并共同转运到目标位点

[38]

MS2/MCP

TIM50TIM22ATP3 mRNA

酵母

通过增加核编码mRNA定位在线粒体表面的数量,响应细胞能量需求的变化

[61]

剪接

MS2/MCP和boxB/λN22

HBB前mRNA

HEK-293细胞

转录会限制剪接的速率

[62]

MS2/MCP和PP7/PCP

RAB7A 前mRNA

人类细胞

剪接体随机选择剪接位点,通过多步过程去除内含子

[63]

mRNA翻译

MS2/MCP

Twist mRNA

果蝇

mRNA翻译效率具有空间异质性

[64]

MS2/MCP

APP mRNA

人神经母细胞瘤细胞

FMRPhnRNP C调控APP mRNA 翻译

[65]

表 2  活细胞RNA成像技术的应用实例
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