Jove-Journal of Visualized Experiments
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2019
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IF 1.108
Yafeng Zhang,Xun Liao,Peihua Luo,Kui Peng,Wenyi Ye,Tengxiang Lian,Qibin Ma,Hai Nian
In plant mitochondria, some steady-state transcripts have 5' triphosphate derived from transcription initiation (primary transcripts), while the others contain 5' monophosphate generated post-transcriptionally (processed transcripts). To discriminate between the two types of transcripts, several strategies have been developed, and most of them depend on presence/absence of 5' triphosphate. However, the triphosphate at primary 5' termini is unstable, and it hinders a clear discrimination of the two types of transcripts. To systematically differentiate and map the primary and processed transcripts stably accumulated in maize mitochondrion, we have developed a circular RT-PCR (cRT-PCR)-based strategy by combining cRT-PCR, RNA 5' polyphoshpatase treatment, quantitative RT-PCR (RT-qPCR), and Northern blot. As an improvement, this strategy includes an RNA normalization step to minimize the influence of unstable 5' triphosphate.In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5' polyphosphatase, which converts 5' triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5' polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5' end and is insensitive to 5' polyphosphatase. After normalization, the primary and processed transcripts are discriminated by comparing cRT-PCR and RT-qPCR products obtained from the treated and non-treated RNAs. The transcript termini are determined by cloning and sequencing of the cRT-PCR products, and then verified by Northern blot.By using this strategy, most steady-state transcripts in maize mitochondrion have been determined. Due to the complicated transcript pattern of some mitochondrial genes, a few steady-state transcripts were not differentiated and/or mapped, though they were detected in a Northern blot. We are not sure whether this strategy is suitable to discriminate and map the steady-state transcripts in other plant mitochondria or in plastids.
研究领域:
植物学
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分子生物学
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DOI
PubMed
高影响因子
CELL
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2025
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IF 42.5
Shaoqian Ma,Yantao Hong,Junhan Chen,Jingzhao Xu,Xiaohua Shen
Understanding cell diversification from a common genome in metazoans requires single-cell transcriptional analysis. We introduce single-cell full-length EU-labeled nascent RNA sequencing (scFLUENT-seq), a single-cell nascent RNA sequencing method using brief 10-min metabolic labeling to capture genome-wide transcription. Surprisingly, individual cells—from splenic lymphocytes to pluripotent stem cells—transcribe only ∼0.02%–3.1% of the genome, versus >80% in bulk, revealing limited genome engagement and profound cell-type and cell-to-cell heterogeneity. Intergenic transcription, especially from heterochromatin, is pervasive and stochastic. Promoter-associated antisense and genic transcription rarely co-occur in the same cell. Proximal intergenic transcription involves both gene readthrough and independent initiation, while distal intergenic transcription is largely independent of neighboring genes and correlates with increased transcriptional diversity, a hallmark of cellular plasticity. Although global RNA synthesis and turnover are coupled in bulk, individual mRNA transcription and decay are poorly coordinated in single cells, suggesting noise-buffering mechanisms. Overall, scFLUENT-seq uncovers complex coding and noncoding transcriptional dynamics that underlie single-cell heterogeneity and state transitions.
研究领域:
分子生物学
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基因组学
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发育生物学
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干细胞生物学
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表观遗传学
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DOI
PubMed
高影响因子
EXPERIMENTAL ANIMALS
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2020
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IF 1.574
Yongxia Cheng,Jingchao Li,Chong Wang,Heran Yang,Ying Wang,Tao Zhan,Sufen Guo,Jun Liang,Yuxin Bai,Jianbo Yu,Guibo Liu
Diabetic cardiomyopathy (DCM) is one of the cardiovascular complications of diabetes mellitus independent of hypertension, coronary disease, and other heart diseases. The development of DCM is multifactorial and hard to detect at an early stage. Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1) is emerging as a regulator of DCM, the underlying mechanism of its role in DCM has not been elaborated yet. In this study, we established a mouse DCM model via streptozocin injection as evidenced by cell hypertrophy and cell apoptosis of myocardial tissue, and found that Malat1 expression was upregulated in the myocardium in DCM mice. Meanwhile, elevated expression of pro-apoptotic factors p53, p21, cleaved caspase 3, cleaved caspase 9 and BAX, and down-regulation of anti-apoptotic BCL-2 were observed in DCM myocardium. We further investigated the effect of Malat1 on cardiomyocytes under high glucose condition by silencing Malat1 with its specific short-hairpin RNA. Like in vivo, expression of Malat1 in cardiomyocytes was notably raised, remarkable cell apoptosis and changes in apoptosis-related factors were also observed following high glucose treatment. Besides, we validated that Malat1 acted as a sponge of miR-181a-5p. Inhibition of miR-181a-5p could, at least partially, abolish Malat1 knockdown-induced alteration in cardiomyocytes. In addition, p53, a critical regulator of apoptosis, was validated to be a downstream target of miR-181a-5p. In summary, our findings reveal that Malat1 knockdown attenuates high glucose-induced cardiomyocyte apoptosis via releasing miR-181a-5p, and this mechanism may provide us with new diagnosis target of DCM.
研究领域:
分子生物学
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心脏病学
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糖尿病
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非编码RNA
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DOI
PubMed
高影响因子
NUCLEIC ACIDS RESEARCH
·
2025
·Vol. 53
·期 6
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IF 13.1
Mao Shiqi,Wu Ruonan,Luo Weibang,Qin Jinshan,Chen Antony K
Clustered regularly interspaced short palindromic repeats (CRISPR)-based imaging tools that utilize fluorescently tagged single-guide RNAs (sgRNAs) have enabled versatile analysis of the dynamics of single genomic loci, but the accuracy may be hindered by nonspecific subnuclear probe accumulation, generating false-positive foci in cell nuclei. By examining the subcellular localizations of sgRNA expression plasmids, their RNA transcripts, and several RNA-binding proteins, we found that spuriously transcribed (cryptic) transcripts, produced by sgRNA expression plasmids, are the major contributors of false-positive signals, independent of sgRNA scaffold design or effector probe (i.e. RNA aptamer- or oligonucleotide-based probes) used. These transcripts interact with the paraspeckle core proteins, but not with the sgRNA expression plasmids or the paraspeckle RNA scaffold NEAT1_2, to form nuclear bodies that display liquid-like properties including sphericality, fusion competence, and sensitivity to 1,6-hexanediol. Transfecting sgRNA transcription units (i.e. sgRNA expression cassettes), lacking the plasmid backbones, reduces false-positive signals and enhances genomic imaging accuracy. Overall, this study unveils previously undescribed activities of cryptic plasmid transcripts and presents an easy-to-adapt strategy that can potentially improve the precision of CRISPR-based imaging systems that implement fluorescently tagged sgRNAs.
研究领域:
分子生物学
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遗传学
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生物物理学
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细胞生物学
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DOI
PubMed
高影响因子
TOXICOLOGY
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2021
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IF 4.221
Xiao Y. Li,Ying J. Qin,Yue Wang,Tao Huang,Yuan H. Zhao,Xiao H. Wang,Christopher J. Martyniuk,Bing Yan
Strobilurin fungicides are used globally and have been detected in microgram per liter concentrations in aquatic environments. Here, we determined the potential toxicity of four commonly used strobilurins (azoxystrobin, kresoxim-methyl, pyraclostrobin, trifloxystrobin) on mitochondrial function and locomotor activity of larval zebrafish at an environmentally relevant level. As the mode of action of strobilurins in fungi is binding to cytochrome bc1 in mitochondrial complex III, we evaluated exposure effects on mitochondrial oxidative phosphorylation of zebrafish, by measuring oxygen consumption rates, mitochondria-related enzyme activities , and transcripts levels for genes associated with the electron transfer chain and citric acid cycle. We found that 50 nM pyraclostrobin and trifloxystrobin lowered basal respiration, oligomycin-induced ATP respiration, and maximal respiration of embryos. Dysfunction in mitochondrial bioenergetics was associated with changes in mitochondrial complex III activity and transcripts of oxidative respiration and stress-related genes. Lower activity of complex III, and reduced cytb mRNA levels were hypothesized to contribute to reduced electron supply to complex IV and V. Both coxI and atp6 were up-regulated, suggesting a compensatory response to impaired oxidative respiration. Cluster analysis indicated that strobilurin-induced oxidative stress and cytb transcript were related to impaired oxidative phosphorylation. We also assessed larval behavior responses, as reduced ATP can affect activity. We observed that pyraclostrobin and trifloxystrobin induced hypoactive responses in zebrafish. At 50 nM, azoxystrobin and kresoxim-methyl exerted no effects on mitochondrial function nor locomotion of zebrafish. Studies such as this are important for determining sublethal toxicity to these fungicides, as widespread detection of strobilurins in aquatic environments suggests there is a potential for adverse effects in aquatic organisms .
研究领域:
环境毒理学
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水生毒理学
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分子生物学
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动物学
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DOI
PubMed
高影响因子
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