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An optimized workflow of full-length transcriptome sequencing for accurate fusion transcript identification. 优化全长转录组测序工作流程,准确识别融合转录本。
IF 3.6 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 Epub Date: 2024-11-14 DOI: 10.1080/15476286.2024.2425527
Liang Zong, Yabing Zhu, Yuan Jiang, Ying Xia, Qun Liu, Jing Wang, Song Gao, Bei Luo, Yongxian Yuan, Jingjiao Zhou, Sanjie Jiang

Next-generation sequencing has revolutionized cancer genomics by enabling high-throughput mutation screening yet detecting fusion genes reliably remains challenging. Long-read sequencing offers potential for accurate fusion transcript identification, though challenges persist. In this study, we present an optimized workflow using nanopore sequencing technology to precisely identify fusion transcripts. Our approach encompasses a tailored library preparation protocol, data processing, and fusion gene analysis pipeline. We evaluated the performance using Universal Human Reference RNA and human adenocarcinoma cell lines. Our optimized nanopore sequencing workflow generated high-quality full-length transcriptome data characterized by an extended length distribution and comprehensive transcript coverage. Validation experiments confirmed novel fusion events with potential clinical relevance. Our protocol aims to mitigate biases and enhance accuracy, facilitating increased adoption in clinical diagnostics. Continued advancements in long-read sequencing promise deeper insights into fusion gene biology and improved cancer diagnostics.

下一代测序技术实现了高通量突变筛选,从而彻底改变了癌症基因组学,但可靠地检测融合基因仍是一项挑战。长读测序为准确鉴定融合转录本提供了潜力,但挑战依然存在。在本研究中,我们介绍了一种利用纳米孔测序技术精确鉴定融合转录本的优化工作流程。我们的方法包括量身定制的文库制备方案、数据处理和融合基因分析流水线。我们使用通用人类参考 RNA 和人类腺癌细胞系对其性能进行了评估。我们优化的纳米孔测序工作流程生成了高质量的全长转录组数据,其特点是长度分布更广、转录本覆盖更全面。验证实验证实了具有潜在临床意义的新型融合事件。我们的方案旨在减少偏差并提高准确性,从而促进临床诊断中更多地采用这种方法。长读数测序技术的不断进步有望加深人们对融合基因生物学的认识并改进癌症诊断。
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引用次数: 0
Origin of ribonucleotide recognition motifs through ligand mimicry at early earth. 核糖核苷酸识别图案的起源是早期地球上的配体模仿。
IF 3.6 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 Epub Date: 2024-11-11 DOI: 10.1080/15476286.2024.2423149
Deepto Mozumdar, Raktim N Roy

In an RNA world, the emergence of template-specific self-replication and catalysis necessitated the presence of motifs facilitating reliable recognition between RNA molecules. What did these motifs entail, and how did they evolve into the proteinaceous RNA recognition entities observed today? Direct observation of these primordial entities is hindered by rapid degradation over geological time scales. To overcome this challenge, researchers employ diverse approaches, including scrutiny of conserved sequences and structural motifs across extant organisms and employing directed evolution experiments to generate RNA molecules with specific catalytic abilities. In this review, we delve into the theme of ribonucleotide recognition across key periods of early Earth's evolution. We explore scenarios of RNA interacting with small molecules and examine hypotheses regarding the role of minerals and metal ions in enabling structured ribonucleotide recognition and catalysis. Additionally, we highlight instances of RNA-protein mimicry in interactions with other RNA molecules. We propose a hypothesis where RNA initially recognizes small molecules and metal ions/minerals, with subsequent mimicry by proteins leading to the emergence of proteinaceous RNA binding domains.

在 RNA 世界中,由于出现了特定模板的自我复制和催化反应,因此必须存在一些促进 RNA 分子之间可靠识别的图案。这些图案是什么,它们又是如何进化成今天观察到的蛋白质 RNA 识别实体的呢?对这些原始实体的直接观察受到地质时间尺度上快速退化的阻碍。为了克服这一难题,研究人员采用了多种方法,包括仔细研究现存生物的保守序列和结构基调,以及利用定向进化实验生成具有特定催化能力的 RNA 分子。在本综述中,我们将深入探讨地球早期进化关键时期的核糖核苷酸识别主题。我们探讨了 RNA 与小分子相互作用的情景,并研究了有关矿物质和金属离子在实现结构化核糖核苷酸识别和催化方面作用的假设。此外,我们还强调了 RNA 与其他 RNA 分子相互作用时的 RNA 蛋白拟态实例。我们提出了一种假说,即 RNA 最初识别小分子和金属离子/矿物质,随后蛋白质进行模仿,从而出现了蛋白质 RNA 结合域。
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引用次数: 0
Epitranscriptomic regulation in fasting hearts: implications for cardiac health. 空腹心脏的表转录组调控:对心脏健康的影响。
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 Epub Date: 2024-02-07 DOI: 10.1080/15476286.2024.2307732
Daniel Benak, Kristyna Holzerova, Jaroslav Hrdlicka, Frantisek Kolar, Mark Olsen, Mati Karelson, Marketa Hlavackova

Cardiac tolerance to ischaemia can be increased by dietary interventions such as fasting, which is associated with significant changes in myocardial gene expression. Among the possible mechanisms of how gene expression may be altered are epigenetic modifications of RNA - epitranscriptomics. N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are two of the most prevalent modifications in mRNA. These methylations are reversible and regulated by proteins called writers, erasers, readers, and m6A-repelled proteins. We analysed 33 of these epitranscriptomic regulators in rat hearts after cardioprotective 3-day fasting using RT-qPCR, Western blot, and targeted proteomic analysis. We found that the most of these regulators were changed on mRNA or protein levels in fasting hearts, including up-regulation of both demethylases - FTO and ALKBH5. In accordance, decreased methylation (m6A+m6Am) levels were detected in cardiac total RNA after fasting. We also identified altered methylation levels in Nox4 and Hdac1 transcripts, both of which play a role in the cytoprotective action of ketone bodies produced during fasting. Furthermore, we investigated the impact of inhibiting demethylases ALKBH5 and FTO in adult rat primary cardiomyocytes (AVCMs). Our findings indicate that inhibiting these demethylases reduced the hypoxic tolerance of AVCMs isolated from fasting rats. This study showed that the complex epitranscriptomic machinery around m6A and m6Am modifications is regulated in the fasting hearts and might play an important role in cardiac adaptation to fasting, a well-known cardioprotective intervention.

禁食等饮食干预措施可增强心脏对缺血的耐受性,而禁食与心肌基因表达的显著变化有关。改变基因表达的可能机制包括 RNA 的表观遗传修饰--表转录组学。N6-甲基腺苷(m6A)和 N6,2'-O-二甲基腺苷(m6Am)是 mRNA 中最常见的两种修饰。这些甲基化是可逆的,并由称为写入蛋白、擦除蛋白、读取蛋白和 m6A 清除蛋白的蛋白质调控。我们使用 RT-qPCR、Western 印迹和靶向蛋白质组分析方法分析了大鼠心脏在禁食 3 天后的 33 种表转录调节因子。我们发现,在禁食的心脏中,这些调节因子大多在 mRNA 或蛋白质水平上发生了变化,包括 FTO 和 ALKBH5 这两种去甲基化酶的上调。相应地,禁食后在心脏总核糖核酸中检测到甲基化(m6A+m6Am)水平下降。我们还发现 Nox4 和 Hdac1 转录本中的甲基化水平发生了变化,这两种物质在禁食期间产生的酮体的细胞保护作用中发挥作用。此外,我们还研究了抑制脱甲基酶 ALKBH5 和 FTO 对成年大鼠原代心肌细胞(AVCMs)的影响。我们的研究结果表明,抑制这些去甲基化酶会降低从禁食大鼠中分离出来的原代大鼠心肌细胞的缺氧耐受性。这项研究表明,围绕 m6A 和 m6Am 修饰的复杂表转录组机制在禁食心脏中受到调控,并可能在心脏适应禁食(一种众所周知的心脏保护干预措施)过程中发挥重要作用。
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引用次数: 0
Transcriptional landscape of small non-coding RNAs reveals diversity of categories and functions in molluscs. 小非编码 RNA 的转录景观揭示了软体动物中类别和功能的多样性。
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 Epub Date: 2024-05-01 DOI: 10.1080/15476286.2024.2348893
Songqian Huang, Kazutoshi Yoshitake, Shigeharu Kinoshita, Shuichi Asakawa

Small non-coding RNAs (sncRNAs) are non-coding RNA molecules that play various roles in metazoans. Among the sncRNAs, microRNAs (miRNAs) guide post-translational gene regulation during cellular development, proliferation, apoptosis, and differentiation, while PIWI-interacting RNAs (piRNAs) suppress transposon activity to safeguard the genome from detrimental insertion mutagenesis. While an increasing number of piRNAs are being identified in the soma and germlines of various organisms, they are scarcely reported in molluscs. To unravel the small RNA (sRNA) expression patterns and genomic function in molluscs, we generated a comprehensive sRNA dataset by sRNA sequencing (sRNA-seq) of eight mollusc species. Abundant miRNAs were identified and characterized in all investigated molluscs, and ubiquitous piRNAs were discovered in both somatic and gonadal tissues in six of the investigated molluscs, which are more closely associated with transposon silencing. Tens of piRNA clusters were also identified based on the genomic mapping results, which varied among different tissues and species. Our dataset serves as important reference data for future genomic and genetic studies on sRNAs in these molluscs and related species, especially in elucidating the ancestral state of piRNAs in bilaterians.

小非编码 RNA(sncRNA)是一种非编码 RNA 分子,在后生动物中发挥着各种作用。在 sncRNAs 中,微小 RNAs(miRNAs)在细胞发育、增殖、凋亡和分化过程中指导翻译后基因调控,而 PIWI 交互作用 RNAs(piRNAs)则抑制转座子活性,保护基因组免受有害插入突变的影响。虽然在各种生物的体细胞和种系中发现了越来越多的 piRNA,但在软体动物中却鲜有报道。为了揭示软体动物体内小 RNA(sRNA)的表达模式和基因组功能,我们通过对 8 种软体动物进行 sRNA 测序(sRNA-seq)生成了一个全面的 sRNA 数据集。在所有被研究的软体动物中,我们发现了丰富的 miRNAs,并对其进行了表征;在其中六种软体动物的体细胞和性腺组织中,我们发现了无处不在的 piRNAs,它们与转座子沉默的关系更为密切。根据基因组图谱结果还发现了数十个 piRNA 簇,这些 piRNA 簇在不同组织和物种中各不相同。我们的数据集可作为今后对这些软体动物及相关物种的 sRNAs 进行基因组学和遗传学研究的重要参考数据,特别是在阐明两栖动物中 piRNAs 的祖先状态方面。
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引用次数: 0
Circular RNAs in non-alcoholic fatty liver disease: Functions and clinical significance. 非酒精性脂肪肝中的环状 RNA:功能和临床意义
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 Epub Date: 2023-12-19 DOI: 10.1080/15476286.2023.2290769
Qingmin Zeng, Chang-Hai Liu, Javier Ampuero, Dongbo Wu, Wei Jiang, Lingyun Zhou, Hong Li, Lang Bai, Manuel Romero-Gómez, Hong Tang

Nonalcoholic fatty liver disease (NAFLD), which affects approximately 25% of the global population, is an urgent health issue leading to various metabolic comorbidities. Circular RNAs (circRNAs), covalently closed RNA molecules, are characterized by ubiquity, diversity, stability, and conservatism. Indeed, they participate in various biological processes via distinct mechanisms that could modify the natural history of NAFLD. In this review, we briefly introduce the biogenesis, characteristics, and biological functions of circRNAs. Furthermore, we summarize circRNAs expression profiles in NAFLD by intersecting seven sequencing data sets and describe the cellular roles of circRNAs and their potential advantages as biomarkers of NAFLD. In addition, we emphatically discuss the exosomal non-coding RNA sorting mechanisms and possible functions in recipient cells. Finally, we extensively discuss the potential application of targeting disease-related circRNAs and competing endogenous RNA networks through gain-of-function and loss-of-function approaches in targeted therapy of NAFLD.

非酒精性脂肪肝(NAFLD)影响着全球约 25% 的人口,是一个亟待解决的健康问题,会导致各种代谢并发症。环状 RNA(circRNA)是共价封闭的 RNA 分子,具有普遍性、多样性、稳定性和保守性等特点。事实上,它们通过不同的机制参与各种生物过程,可改变非酒精性脂肪肝的自然病史。在这篇综述中,我们简要介绍了 circRNA 的生物发生、特点和生物学功能。此外,我们还通过交叉七组测序数据总结了非酒精性脂肪肝中 circRNAs 的表达谱,并描述了 circRNAs 的细胞作用及其作为非酒精性脂肪肝生物标志物的潜在优势。此外,我们还着重讨论了外泌体非编码 RNA 的分选机制以及在受体细胞中可能发挥的功能。最后,我们广泛讨论了在非酒精性脂肪肝的靶向治疗中,通过功能增益和功能缺失方法靶向疾病相关的 circRNA 和竞争性内源性 RNA 网络的潜在应用。
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引用次数: 0
Structure and function of the pseudouridine 5'-monophosphate glycosylase PUMY from Arabidopsis thaliana. 拟南芥中假尿苷-5'-单磷酸糖基化酶 PUMY 的结构和功能。
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 Epub Date: 2023-12-20 DOI: 10.1080/15476286.2023.2293340
Jeongyun Lee, Sang-Hoon Kim, Sangkee Rhee

Pseudouridine is a noncanonical C-nucleoside containing a C-C glycosidic linkage between uracil and ribose. In the two-step degradation of pseudouridine, pseudouridine 5'-monophosphate glycosylase (PUMY) is responsible for the second step and catalyses the cleavage of the C-C glycosidic bond in pseudouridine 5'-monophosphate (ΨMP) into uridine and ribose 5'-phosphate, which are recycled via other metabolic pathways. Structural features of Escherichia coli PUMY have been reported, but the details of the substrate specificity of ΨMP were unknown. Here, we present three crystal structures of Arabidopsis thaliana PUMY in different ligation states and a kinetic analysis of ΨMP degradation. The results indicate that Thr149 and Asn308, which are conserved in the PUMY family, are structural determinants for recognizing the nucleobase of ΨMP. The distinct binding modes of ΨMP and ribose 5'-phosphate also suggest that the nucleobase, rather than the phosphate group, of ΨMP dictates the substrate-binding mode. An open-to-close transition of the active site is essential for catalysis, which is mediated by two α-helices, α11 and α12, near the active site. Mutational analysis validates the proposed roles of the active site residues in catalysis. Our structural and functional analyses provide further insight into the enzymatic features of PUMY towards ΨMP.

伪尿嘧啶是一种非典型的 C 核苷,含有尿嘧啶和核糖之间的 C-C 糖苷键。在假尿苷的两步降解过程中,假尿嘧啶-5'-单磷酸糖基化酶(PUMY)负责第二步,催化假尿嘧啶-5'-单磷酸(ΨMP)中的 C-C 糖苷键裂解为尿苷和核糖-5'-磷酸,并通过其他代谢途径循环利用。已有关于大肠杆菌 PUMY 结构特征的报道,但ΨMP 底物特异性的细节尚不清楚。在这里,我们展示了拟南芥 PUMY 在不同连接状态下的三个晶体结构以及ΨMP 降解的动力学分析。结果表明,在 PUMY 家族中保守的 Thr149 和 Asn308 是识别 ΨMP 核碱基的结构决定因素。ΨMP与核糖-5'-磷酸的不同结合模式也表明,ΨMP的核碱基而不是磷酸基决定了底物的结合模式。活性位点从开放到封闭的转变是催化所必需的,这种转变是由活性位点附近的两个α-螺旋(α11 和 α12)介导的。突变分析验证了活性位点残基在催化作用中的作用。我们的结构和功能分析进一步揭示了 PUMY 对 ΨMP 的酶学特征。
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引用次数: 0
Ordering events in a developing genetic code. 发育中遗传密码的排序事件
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 Epub Date: 2024-01-03 DOI: 10.1080/15476286.2023.2299615
Michael Yarus

Preexisting partial genetic codes can fuse to evolve towards the complete Standard Genetic Code (SGC). Such code fusion provides a path of 'least selection', readily generating precursor codes that resemble the SGC. Consequently, such least selections produce the SGC via minimal, thus rapid, change. Optimal code evolution therefore requires delayed wobble. Early wobble encoding slows code evolution, very specifically diminishing the most likely SGC precursors: near-complete, accurate codes which are the products of code fusions. In contrast: given delayed wobble, the SGC can emerge from a truncation selection/evolutionary radiation based on proficient fused coding.

已有的部分遗传密码可以融合进化成完整的标准遗传密码(SGC)。这种代码融合提供了一种 "最少选择 "的途径,很容易产生与 SGC 相似的前体代码。因此,这种 "最少选择 "可以通过最小的变化,也就是快速变化产生 SGC。因此,最佳的代码进化需要延迟的摇摆。早期摇摆编码会减慢代码进化的速度,特别是会减少最有可能的 SGC 前体:近乎完整、准确的代码,它们是代码融合的产物。与此相反的是:在延迟摇摆的情况下,SGC 可以从基于熟练融合编码的截断选择/进化辐射中产生。
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引用次数: 0
Small RNA-big impact: exosomal miRNAs in mitochondrial dysfunction in various diseases. 小 RNA 大影响:外泌体 miRNA 在各种疾病的线粒体功能障碍中的作用。
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 Epub Date: 2024-01-04 DOI: 10.1080/15476286.2023.2293343
Xiaqing Li, Yi Han, Yu Meng, Lianghong Yin

Mitochondria are multitasking organelles involved in maintaining the cell homoeostasis. Beyond its well-established role in cellular bioenergetics, mitochondria also function as signal organelles to propagate various cellular outcomes. However, mitochondria have a self-destructive arsenal of factors driving the development of diseases caused by mitochondrial dysfunction. Extracellular vesicles (EVs), a heterogeneous group of membranous nano-sized vesicles, are present in a variety of bodily fluids. EVs serve as mediators for intercellular interaction. Exosomes are a class of small EVs (30-100 nm) released by most cells. Exosomes carry various cargo including microRNAs (miRNAs), a class of short noncoding RNAs. Recent studies have closely associated exosomal miRNAs with various human diseases, including diseases caused by mitochondrial dysfunction, which are a group of complex multifactorial diseases and have not been comprehensively described. In this review, we first briefly introduce the characteristics of EVs. Then, we focus on possible mechanisms regarding exosome-mitochondria interaction through integrating signalling networks. Moreover, we summarize recent advances in the knowledge of the role of exosomal miRNAs in various diseases, describing how mitochondria are changed in disease status. Finally, we propose future research directions to provide a novel therapeutic strategy that could slow the disease progress mediated by mitochondrial dysfunction.

线粒体是参与维持细胞平衡的多任务细胞器。线粒体除了在细胞生物能方面发挥公认的作用外,还作为信号细胞器传播各种细胞结果。然而,线粒体也有自我毁灭的因素,这些因素导致了线粒体功能障碍疾病的发生。细胞外小泡(EVs)是一类异构的膜状纳米级小泡,存在于各种体液中。细胞外小泡是细胞间相互作用的媒介。外泌体是由大多数细胞释放的一类小型 EV(30-100 纳米)。外泌体携带各种货物,包括微小核糖核酸(miRNA),这是一类短的非编码核糖核酸。最近的研究发现,外泌体 miRNA 与多种人类疾病密切相关,其中包括线粒体功能障碍引起的疾病,这些疾病是一组复杂的多因素疾病,尚未得到全面描述。在这篇综述中,我们首先简要介绍了 EVs 的特征。然后,我们重点讨论了外泌体-线粒体通过整合信号网络相互作用的可能机制。此外,我们还总结了外泌体 miRNAs 在各种疾病中作用的最新进展,描述了线粒体在疾病状态下的变化。最后,我们提出了未来的研究方向,以提供一种新的治疗策略,减缓由线粒体功能障碍介导的疾病进展。
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引用次数: 0
An elusive debate on the evidence for RNA editing in SARS-CoV-2. 关于 SARS-CoV-2 中 RNA 编辑证据的一场难以捉摸的辩论。
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 Epub Date: 2024-03-01 DOI: 10.1080/15476286.2024.2321032
Giorgio Mattiuz, Salvatore Di Giorgio, Silvestro G Conticello
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引用次数: 0
The role of the 5' sensing function of ribonuclease E in cyanobacteria. 蓝藻中核糖核酸酶 E 的 5'感应功能的作用。
IF 3.6 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 Epub Date: 2024-03-12 DOI: 10.1080/15476286.2024.2328438
Ute A Hoffmann, Elisabeth Lichtenberg, Said N Rogh, Raphael Bilger, Viktoria Reimann, Florian Heyl, Rolf Backofen, Claudia Steglich, Wolfgang R Hess, Annegret Wilde

RNA degradation is critical for synchronising gene expression with changing conditions in prokaryotic and eukaryotic organisms. In bacteria, the preference of the central ribonucleases RNase E, RNase J and RNase Y for 5'-monophosphorylated RNAs is considered important for RNA degradation. For RNase E, the underlying mechanism is termed 5' sensing, contrasting to the alternative 'direct entry' mode, which is independent of monophosphorylated 5' ends. Cyanobacteria, such as Synechocystis sp. PCC 6803 (Synechocystis), encode RNase E and RNase J homologues. Here, we constructed a Synechocystis strain lacking the 5' sensing function of RNase E and mapped on a transcriptome-wide level 283 5'-sensing-dependent cleavage sites. These included so far unknown targets such as mRNAs encoding proteins related to energy metabolism and carbon fixation. The 5' sensing function of cyanobacterial RNase E is important for the maturation of rRNA and several tRNAs, including tRNAGluUUC. This tRNA activates glutamate for tetrapyrrole biosynthesis in plant chloroplasts and in most prokaryotes. Furthermore, we found that increased RNase activities lead to a higher copy number of the major Synechocystis plasmids pSYSA and pSYSM. These results provide a first step towards understanding the importance of the different target mechanisms of RNase E outside Escherichia coli.

在原核生物和真核生物中,RNA 降解对于使基因表达与不断变化的条件同步至关重要。在细菌中,中心核糖核酸酶 RNase E、RNase J 和 RNase Y 对 5'-monophosphorylated RNA 的偏好被认为是 RNA 降解的重要因素。对于 RNase E 来说,其基本机制被称为 "5'末端感应"(5's sensing),这与另一种 "直接进入"(direct entry)模式不同,后者与单磷酸化的 5'末端无关。蓝藻,如 Synechocystis sp. PCC 6803(Synechocystis),编码 RNase E 和 RNase J 同源物。在这里,我们构建了一个缺乏 RNase E 的 5'感应功能的 Synechocystis 菌株,并在整个转录组水平上绘制了 283 个依赖于 5'感应的裂解位点。这些位点包括迄今未知的目标,如编码与能量代谢和碳固定有关的蛋白质的 mRNA。蓝藻 RNase E 的 5'感应功能对 rRNA 和几种 tRNA(包括 tRNAGluUUC)的成熟非常重要。在植物叶绿体和大多数原核生物中,这种 tRNA 可激活谷氨酸进行四吡咯的生物合成。此外,我们还发现 RNase 活性的增加会导致 Synechocystis 主要质粒 pSYSA 和 pSYSM 的拷贝数增加。这些结果为了解大肠杆菌外 RNase E 不同靶机制的重要性迈出了第一步。
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引用次数: 0
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