Chemical Science
·
2021
·Vol. 12
·期 23
·8149-8156
·
IF 9.825
Ming-Yu Cheng,Xue-Jiao You,Jiang-Hui Ding,Yi Dai,Meng-Yuan Chen,Bi-Feng Yuan,Yu-Qi Feng
RNA modifications play critical roles in regulating a variety of physiological processes. Methylation is the most prevalent modification occurring in RNA. Three isomeric cytidine methylation modifications have been reported in RNA, including 3-methylcytidine (m3C), N4-methylcytidine (m4C), and 5-methylcytidine (m5C), in mammals. Aside from the single methylation on the nucleobase of cytidines, dual methylation modifications occurring in both the 2′ hydroxyl of ribose and the nucleobase of cytidines also have been reported, including N4,2′-O-dimethylcytidine (m4Cm) and 5,2′-O-dimethylcytidine (m5Cm). m4Cm has been found in the 16S rRNA of E. coli, while m5Cm has been found in the tRNA of terminal thermophilic archaea and mammals. However, unlike m4Cm and m5Cm, the presumed dual methylation of 3,2′-O-dimethylcytidine (m3Cm) has never been discovered in living organisms. Thus, the presence of m3Cm in RNA remains an open question. In the current study, we synthesized m3Cm and established a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to determine the dimethylation of cytidines, m3Cm, m4Cm and m5Cm. Under optimized analytical conditions, m3Cm, m4Cm and m5Cm can be clearly distinguished. Using the method, we discovered the existence of m3Cm in the RNA of mammals. The identified m3Cm is a novel modification that hasn't been reported in the three-domain system, including archaea, bacteria, and eukaryotes. We confirmed that m3Cm mainly existed in the small RNA (<200 nt) of mammals. In addition, we identified, for the first time, the presence of m4Cm in the 18S rRNA of mammalian cells. The stable isotope tracing monitored by mass spectrometry demonstrated that S-adenosyl-L-methionine was a methyl donor for all three dimethylations of cytidines in RNA. The discovery of m3Cm broadens the diversity of RNA modifications in living organisms. In addition, the discovery of m3Cm and m4Cm in mammals opens new directions in understanding RNA modification-mediated RNA processing and gene expression regulation.
研究领域:
分子生物学
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生物化学
·
RNA生物学
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DOI
PubMed
高影响因子
PHOTOSYNTHESIS RESEARCH
·
2015
·Vol. 126
·期 2
·311-321
·
IF 3.502
Zhang Hong-Dao,Cui Yong-Lan,Huang Chao,Yin Qian-Qian,Qin Xue-Mei,Xu Te,He Xiao-Fang,Zhang Yi,Li Zi-Ran,Yang Zhong-Nan
After transcription, most chloroplast precursor RNAs undergo further post-transcriptional processing including cleavage, editing, and splicing. Previous investigation has shown that the cleavage of the rpoA transcript and most editing sites, including accD - 1 , are defective in the knockout mutant of PDM1/SEL1, a PLS-type PPR protein, and that PDM1 is associated with the rpoA transcript. In this work, we found that the splicing of group II introns in trnK and ndhA is also affected in pdm1 . Co-immunoprecipitation mass spectrometry experiments were performed to identify proteins that are associated with PDM1. We obtained 126 non-redundant proteins, of which MORF9 was reported to be involved in RNA editing in chloroplast. Yeast two-hybrid assays showed that PDM1 interacts directly with MORF9, MORF2, and MORF8. RNA immunoprecipitation showed that PDM1 associates with the transcripts of trnK and ndhA , as well as accD - 1, suggesting that PDM1 is involved in RNA editing and splicing. Therefore, PDM1 is an important protein for post-transcriptional regulation in chloroplast.
研究领域:
植物学
·
分子生物学
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DOI
PubMed
高影响因子
Chemical Science
·
2020
·Vol. 11
·期 7
·1878-1891
·
IF 9.346
Qing-Yun Cheng,Jun Xiong,Cheng-Jie Ma,Yi Dai,Jiang-Hui Ding,Fei-Long Liu,Bi-Feng Yuan,Yu-Qi Feng
The discovery of dynamic and reversible modifications in messenger RNA (mRNA) is opening new directions in RNA modification-mediated regulation of biological processes. Methylation is the most prevalent modification occurring in mRNA and the methyl group is mainly decorated in the adenine, cytosine, and guanine base or in the 2′-hydroxyl group of ribose. However, methylation of the uracil base (5-methyluridine, m5U) has not been discovered in mRNA of eukaryotes. In the current study, we established a method of N-cyclohexyl-N′-β-(4-methylmorpholinium) ethylcarbodiimide p-toluenesulfonate (CMCT) labelling coupled with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) analysis for the sensitive determination of uridine modifications in RNA. Our results demonstrated that the detection sensitivities of uridine modifications in RNA increased up to 1408 fold upon CMCT labelling. Using the developed method, we identified the distinct existence of m5U in mRNA of various mammalian cells and tissues. In addition, the stable isotope tracing monitored by mass spectrometry revealed that the methyl group of m5U originated from S-adenosyl-L-methionine (SAM). Our study expanded the list of modifications occurring in mRNA of mammals. Future work on transcriptome-wide mapping of m5U will further uncover the functional roles of m5U in mRNA of mammals.
研究领域:
分子生物学
·
生物化学
·
表观遗传学
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产品关联
DOI
PubMed
高影响因子
Nature Communications
·
2025
·Vol. 16
·期 1
·1-15
·
IF 15.7
Chen Zhenyin,Chen Wei,Xu Cun,Song Haozhi,Ji Xin,Jiang Haodong,Duan Hongtao,Li Zehao,Gao Wankai,Yao Tuoxin,Zhang Zhongxuan,He Liuqin,Yin Yulong,Yang Nanyang,Tian Wenjing,Wu Jiahui,Li Xing
Fluorogenic RNA aptamers have various applications, including use as fluorescent tags for imaging RNA trafficking and as indicators of RNA-based sensors that exhibit fluorescence upon binding small-molecule fluorophores in living cells. Current fluorogenic RNA:fluorophore complexes typically emit visible fluorescence. However, it is challenging to develop fluorogenic RNA with near-infrared (NIR) fluorescence for in vivo imaging and sensing studies. To address this issue, we identify and modulate red fluorescent protein-like fluorophores to bind Squash, a highly folded fluorogenic RNA. One of these fluorophores, DFQL-1T, exhibits photostable NIR fluorescence when bound to Squash, enabling RNA visualization in living mammalian cells and mice. With Squash:DFQL-1T complexes, we generate RNA-based sensors for detecting non-coding RNAs and small molecule targets in living mammalian cells and in mice. These studies reveal a fluorogenic RNA:fluorophore complex that can be readily developed into NIR fluorescent RNA tags for in vivo imaging and sensing.
研究领域:
分子生物学
·
生物医学工程
·
成像科学
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产品关联
DOI
PubMed
高影响因子
STAR Protocols
·
2022
·
IF 0
Yajie Gao,Yuanxiang Zhu,Qinmiao Sun,Dahua Chen
Summary Here, we provide an optimized RNA-induced silencing complex (RISC) assembly and cleavage protocol in vitro without using radiolabeled RNA. The protocol is useful to characterize the biochemical properties of the RISC. We describe the preparation of RNA probes, the target RNA, and Drosophila cell lysates for RISC assembly assay. We then detail AGO1 complexes immunoprecipitation for RISC cleavage assay. This protocol can detect RISC assembly and cleavage products within 5 days. Moreover, it can detect 5′- and 3′-cleavage products simultaneously. For complete details on the use and execution of this protocol, please refer to Gao et al. (2022) .
研究领域:
分子生物学
·
生物化学
·
细胞生物学
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DOI
PubMed
Cell Reports
·
2025
·
IF 6.9
Tianzi Wei,Jiaxuan Li,Xiang Lei,Risheng Lin,Qingyan Wu,Zhenfeng Zhang,Shimin Shuai,Ruilin Tian
Adenosine-to-inosine (A-to-I) RNA editing is a critical post-transcriptional modification that diversifies the transcriptome and influences various cellular processes, yet its regulatory mechanisms remain largely unknown. Here, we present two complementary CRISPR-based genetic screening platforms: CREDITS (CRISPR-based RNA editing regulator screening), which enables genome-scale identification of editing regulators using an RNA recorder-based reporter system, and scCREDIT-seq (single-cell CRISPR-based RNA editing sequencing), which provides multiplexed single-cell characterization of transcriptome and editome changes for pooled perturbations. By screening 1,350 RNA-binding proteins, we identified a series of A-to-I editing regulators. Mechanistic investigation revealed DDX39B as a global repressor of A-to-I editing, which functions by preventing double-stranded RNA accumulation through its helicase activity. Targeting DDX39B significantly enhances the efficiency of RNA-editing-based tools, such as CellREADR (cell access through RNA sensing by endogenous ADAR) and LEAPER (leveraging endogenous ADAR for programmable editing of RNA), and disrupts hepatitis D virus (HDV) RNA editing homeostasis. These technological advances not only expand our understanding of RNA editing regulation but also provide powerful tools for exploring tissue-specific and context-dependent RNA modification mechanisms, with broad implications for therapeutic development.
研究领域:
分子生物学
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遗传学
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RNA生物学
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CRISPR技术
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病毒学
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基因编辑
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功能基因组学
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产品关联
DOI
PubMed
高影响因子
TALANTA
·
2019
·
IF 4.916
Zengjin Li,Hui Guo,Fangyuan Xu,Wei Tang,Xinrui Duan
We reported a new strategy for sensitive monitoring in vitro RNA synthesis in real time based on fluorescence resonance energy transfer (FRET) from water-soluble conjugated polymer poly (9, 9-bis (6′-N, N, N,-trimethylammonium) hexyl) fluorene-co-alt-1,4-phenylene) bromide (PFP) to fluorogenic RNA aptamer/fluorophore (Spanich2/DFHBI and Broccoli/DFHBI) system. In this strategy, RNA of interest was transcribed accompanied by the Spanich2 or Broccoli. Then the 3, 5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) bound to the RNA aptamer sequence and thereby induced a fluorescence signal. PFP was used as the fluorescence energy donor, and Spanich2/DFHBI was the fluorescence energy acceptor. The fluorescence signal of Spanich2/DFHBI was amplified by light-harvesting and fluorescence amplification ability of PFP via FRET. And the limit of detection (LOD) (0.29 nM) was near 10-fold lower than that of RNA aptamer/DFHBI (LOD is 2.8 nM) alone by measuring the FRET ratio, which greatly reduced the variation of background signals. Most importantly, the addition of PFP did not interfere with RNA transcription in vitro , so this method was successfully applied to sensitively monitor RNA transcription and effect of T7 RNA polymerase inhibitor in real time, supplying a sensitive and simple method to study the modulation and inhibitor of RNA polymerase in vitro .
研究领域:
生物化学
·
分子生物学
·
分析化学
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产品关联
DOI
PubMed
高影响因子
ACS Synthetic Biology
·
2022
·
IF 5.249
Dongying Wei,Yueyao Wang,Dongfan Song,Ze Zhang,Juan Wang,Jia-Yu Chen,Zhe Li,Hanyang Yu
Threose nucleic acid (TNA) is considered a potential RNA progenitor due to its chemical simplicity, base pairing property, and capability of folding into a functional tertiary structure. However, it is unknown whether the functional property can be maintained during transition from TNA to RNA. Here, we use a toggle in vitro selection to identify nucleic acid catalyst sequences that are active in both TNA and RNA backbones. One such nucleic acid enzyme with exchangeable backbone (CAMELEON) catalyzes an RNA cleavage reaction when prepared as TNA (T) and RNA (R). Further biochemical characterization reveals that CAMELEON R and T exhibit different catalytic behaviors such as rate enhancement and magnesium dependence. Structural probing and mutagenesis experiments suggest that they likely fold into distinct tertiary structures. This work demonstrates that the catalytic activity can be preserved during backbone transition from TNA to RNA and provides further experimental support for TNA as an RNA precursor in evolution.
研究领域:
生物化学
·
分子进化
·
核酸化学
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DOI
PubMed
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2020
·Vol. 412
·期 26
·7349-7356
·
IF 3.637
Li Jun,Wang Man,Fan Yao-Yao,Duan Hui-Ling,Deng Xu,Zhang Zhi-Qi
The application of the dye-labeled fluorescence method in a ligand-RNA interaction assay is a complex and costly process prone to steric hindrance. Fluorescent nanomaterials offer an attractive alternative due to their simple, low-cost synthesis and effective screening properties. Here, CdTe@ZIF-8 core-shell nanocomposites were used as fluorescence signal transducer in the ligand-TAR RNA interaction assay. Different experimental strategies were developed based on the size-selective nature of the CdTe@ZIF-8 nanocomposites. When ligands can quench fluorescence, two assays of fluorescence recovery with TAR RNA and Tat peptide competitive displacement are carried out successively, which can not only distinguish ligands binding to TAR RNA but also screen potential Tat protein antagonists. When ligands cannot quench fluorescence, the mitoxantrone-TAR RNA complex is used in the competitive displacement assay. Ligands that displaced mitoxantrone from the mitoxantrone-TAR RNA complex signaled the interaction with TAR RNA. Eight ligands, including known and unknown TAR RNA-binding ligands, were tested via the above strategies. The results showed that this method was effective at distinguishing the known RNA-binding partner and screening the Tat antagonist from the test ligands. This simple and effective strategy is expected to be suitable for actual drug screening.
Graphical abstract
研究领域:
生物化学
·
分子生物学
·
纳米技术
·
药理学
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产品关联
DOI
PubMed
高影响因子
Current Protocols
·
2024
·Vol. 4
·期 1
·e956
·
IF 0
Xuan Chen,Long Xie,Chenglong Zhang,Shen Tian,Zeyu Tang,Ming Xiang,Weiwei Tian,Patrick Lu,Xianbin Yang
The integration of fluorine atoms into biologically active organic compounds has proved to be a vital technique in small molecule drugs. This technique can substantially enhance crucial properties, including metabolic stability, lipophilicity, and bioavailability, often with a mere addition of a single fluorine atom or a trifluoromethyl group. Over the past few decades, this concept has also been applied in nucleic acid chemistry. A commonly employed 2′-OH substitution is the introduction of a 2′-deoxy-2′-fluoro (2′-F) group. The strong electronegativity of fluorine prompts the modified siRNA to readily adopt a C3′- endo conformation, resulting in significant advantages in terms of binding affinity. To enrich the toolbox of chemical modification of oligonucleotides, the replacement of the 2′-OH with the 2′- O -trifluoromethyl group has been developed in RNA analog synthesis. Oligodeoxynucleotides containing the 2′- O -trifluoromethyl group can greatly increase the thermal stability of DNA/RNA duplexes depending on the position and amount of the modification. Moreover, 2′- O -trifluoromethylated oligodeoxynucleotide also exhibited a slightly higher resistance to snake venom phosphodiesterase than the unmodified oligodeoxynucleotide. The 2′- O -trifluoromethylated oligonucleotides can emerge as a label to study RNA structure and function as well, or to develop DNA/RNA-based diagnostics. Hence, it is necessary to report an effective method for the synthesis, deprotection, purification, and characterization of oligonucleotides bearing a 2′ -O -trifluoromethyl group. © 2024 Wiley Periodicals LLC. Basic Protocol 1 : Preparation of 6- N -benzoyl-5′- O -dimethoxytrityl-2′- O -trifluoromethyl adenosine 3′-(2-cyanoethyl N,N -diisopropyl)phosphoramidite Basic Protocol 2 : Preparation of 4- N -acetyl-5′- O -dimethoxytrityl-2′- O -trifluoromethyl cytidine 3′-(2-cyanoethyl N,N -diisopropyl)phosphoramidite Basic Protocol 3 : Preparation of 2- N -isobutyryl-5′- O -dimethoxytrityl-2′- O -trifluoromethyl guanine 3′-(2-cyanoethyl N,N -diisopropyl)phosphoramidite Basic Protocol 4 : Preparation of 5′- O -dimethoxytrityl-2′- O -2-trifluoromethyl uridine 3′-(2-cyanoethyl N,N -diisopropyl) phosphoramidite Basic Protocol 5 : Solid-phase synthesis of 2′- O -trifluoromethylated RNA analogs Basic Protocol 6 : Deprotection and purification of 2′- O -trifluoromethyl-RNAs
研究领域:
生物有机化学
·
核酸化学
·
药物化学
·
RNA生物学
·
分子生物学
·
药学
✓ 已验证
DOI
Nature Communications
·
2023
·Vol. 14
·期 1
·1-12
·
IF 16.6
Xu Ziye,Zhang Tianyu,Chen Hongyu,Zhu Yuyi,Lv Yuexiao,Zhang Shunji,Chen Jiaye,Chen Haide,Yang Lili,Jiang Weiqin,Ni Shengyu,Lu Fangru,Wang Zhaolun,Yang Hao,Dong Ling,Chen Feng,Zhang Hong,Chen Yu,Liu Jiong,Zhang Dandan,Fan Longjiang,Guo Guoji,Wang Yongcheng
Formalin-fixed paraffin-embedded (FFPE) tissues constitute a vast and valuable patient material bank for clinical history and follow-up data. It is still challenging to achieve single cell/nucleus RNA (sc/snRNA) profile in FFPE tissues. Here, we develop a droplet-based snRNA sequencing technology (snRandom-seq) for FFPE tissues by capturing full-length total RNAs with random primers. snRandom-seq shows a minor doublet rate (0.3%), a much higher RNA coverage, and detects more non-coding RNAs and nascent RNAs, compared with state-of-art high-throughput scRNA-seq technologies. snRandom-seq detects a median of >3000 genes per nucleus and identifies 25 typical cell types. Moreover, we apply snRandom-seq on a clinical FFPE human liver cancer specimen and reveal an interesting subpopulation of nuclei with high proliferative activity. Our method provides a powerful snRNA-seq platform for clinical FFPE specimens and promises enormous applications in biomedical research.
研究领域:
分子生物学
·
基因组学
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癌症研究
·
转录组学
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产品关联
DOI
PubMed
高影响因子
NUCLEIC ACIDS RESEARCH
·
2025
·Vol. 53
·期 2
·
IF 13.1
Zhang Lihang,Qiu Xinyuan,Zhou Yuting,Luo Zhengyang,Zhu Lingyun,Shao Jiawei,Xie Mingqi,Wang Hui
The CRISPR-derived endoribonuclease Csy4 is a popular tool for controlling transgene expression in various therapeutically relevant settings, but adverse effects potentially arising from non-specific RNA cleavage remains largely unexplored. Here, we report a split-Csy4 architecture that was carefully optimized for in vivo usage. First, we separated Csy4 into two independent protein moieties whose full catalytic activity can be restored via various constitutive or conditional protein dimerization systems. Next, we show that introduction of split-Csy4 into human cells caused a substantially reduced extent in perturbation of the endogenous transcriptome when directly compared to full-length Csy4. Inspired by these results, we went on to use such split-Csy4 module to engineer inducible CRISPR- and translation-level gene switches regulated by the FDA-approved drug grazoprevir. This work provides valuable resource for Csy4-related biomedical research and discusses important issues for the development of clinically eligible regulation tools.
研究领域:
分子生物学
·
基因工程
·
治疗学
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）