Animals
·
2025
·Vol. 15
·期 6
·865
·
IF 2.7
Chengcheng Wu,Lihan Tao,Quanyong Zhou,Fanfan Zhang,Yanbing Zeng
Simple SummaryThe helicase NSP13, encoded by porcine deltacoronavirus (PDCoV), regulates viral replication and is considered to bean ideal target for antiviral drugs. In our work, the wild-type NSP13WTand various deletion mutants were generated through expression and purification, and we tested the activities of these proteins using multiple methods. The results revealed that both the Stalk and 1B domains were necessary for NSP13WTto separate the double-stranded nucleic acids, and the deletion of the ZBD impaired this unwinding activity. Our study is the first to explore the role of each domain in the activities of PDCoV NSP13, providing mechanistic insights into helicase-regulated viral replication and laying the foundation for the development of antiviral drugs.The nonstructural protein 13 (NSP13) of PDCoV is a highly conservative helicase and plays key roles in viral replication. NSP13 contains a zinc-binding domain (ZBD), a helical Stalk domain, a beta-barrel 1B domain, and a core helicase domain. However, the specific functions of these domains of PDCoV NSP13 remain largely unknown. Here, we expressed and purified the wild-type NSP13WTand various mutants with domain deletions, and the activities of these proteins were analyzed using multiple methods. We found that NSP13ΔZBDpossessed the abilities to hydrolyze ATP and unwind double-stranded nucleic acids, but the unwinding efficiency was lower than that of NSP13WT. In contrast, NSP13ΔZBD-Stalk, NSP13Δ1B, and NSP13ΔZBD-Stalk-1Ball lost their unwinding activity, but not their ATPase activity. These results revealed that the deletion of the ZBD impaired the unwinding activity of PDCoV helicase NSP13, and the Stalk and 1B domains were critical for NSP13 to separate the duplexes. The identification of the roles of each domain in this study was helpful to gain an in-depth understanding of the overall functions of helicase NSP13, providing a theoretical basis for the development of antiviral drugs targeting helicase.
研究领域:
病毒学
·
分子生物学
·
抗病毒药物开发
✓ 已验证
产品关联
DOI
PubMed
高影响因子
NUCLEIC ACIDS RESEARCH
·
2024
·
IF 16.6
Liao Fumeng,Yu Guimei,Zhang Chendi,Liu Zhikun,Li Xuzichao,He Qiuqiu,Yin Hang,Liu Xiang,Li Zhuang,Zhang Heng
Recently, a novel two-gene bacterial defense system against phages, encoding a SIR2 NADase and a HerA ATPase/helicase, has been identified. However, the molecular mechanism of the bacterial SIR2–HerA immune system remains unclear. Here, we determine the cryo-EM structures of SIR2, HerA and their complex from Paenibacillus sp. 453MF in different functional states. The SIR2 proteins oligomerize into a dodecameric ring-shaped structure consisting of two layers of interlocked hexamers, in which each subunit exhibits an auto-inhibited conformation. Distinct from the canonical AAA+ proteins, HerA hexamer alone in this antiphage system adopts a split spiral arrangement, which is stabilized by a unique C-terminal extension. SIR2 and HerA proteins assemble into a ∼1.1 MDa torch-shaped complex to fight against phage infection. Importantly, disruption of the interactions between SIR2 and HerA largely abolishes the antiphage activity. Interestingly, binding alters the oligomer state of SIR2, switching from a dodecamer to a tetradecamer state. The formation of the SIR2–HerA binary complex activates NADase and nuclease activities in SIR2 and ATPase and helicase activities in HerA. Together, our study not only provides a structural basis for the functional communications between SIR2 and HerA proteins, but also unravels a novel concerted antiviral mechanism through NAD+ degradation, ATP hydrolysis, and DNA cleavage.
研究领域:
微生物学
·
结构生物学
·
分子生物学
✓ 已验证
DOI
PubMed
高影响因子
NUCLEIC ACIDS RESEARCH
·
2022
·Vol. 50
·期 16
·9175-9189
·
IF 19.16
Jia Ruirui,Lin Jiamei,You Jin,Li Shi,Shan Ge,Huang Chuan
Stress-responsive genes are lowly transcribed under normal conditions and robustly induced in response to stress. The significant difference between basal and induced transcription indicates that the general transcriptional machinery requires a mechanism to distinguish each transcription state. However, what factors specifically function in basal transcription remains poorly understood. Using a classic model stress-responsive gene (Drosophila MtnA), we found that knockdown of the DEAD-box helicase Hlc resulted in a significant transcription attenuation of MtnA under normal, but not stressed, conditions. Mechanistically, Hlc directly binds to the MtnA locus to maintain the accessibility of chromatin near the transcriptional start site, which allows the recruitment of RNA polymerase II and subsequent MtnA transcription. Using RNA-seq, we then identified plenty of additional stress-responsive genes whose basal transcription was reduced upon knockdown of Hlc. Taken together, these data suggest that Hlc-mediated basal transcription regulation is an essential and widespread mechanism for precise control of stress-responsive genes.
研究领域:
分子生物学
·
转录调控
·
基因表达
✓ 已验证
DOI
PubMed
高影响因子
CELL BIOLOGY INTERNATIONAL
·
2024
·
IF 3.3
Guang Yang,Xin Shi,Meixia Zhang,Kaiwen Wang,Xin Tian,Xiaofeng Wang
Oral squamous cell carcinoma (OSCC) is the most common oral malignancy. DEAD/H-box helicase 11 (DDX11), a DNA helicase, has been implicated in the progression of several cancers. Yet, the precise function of DDX11 in OSCC is poorly understood. The DDX11 expression in OSCC cells and normal oral keratinocytes was evaluated in the Gene Expression Omnibus database (GSE146483 and GSE31853). SCC-4 and CAL-27 cells expressing doxycycline-inducible DDX11 or DDX11 shRNA were generated by lentiviral infection. The role of DDX11 in OSCC cells was determined by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, colony formation assay, flow cytometry assay, TUNEL staining, and western blot. The effects of DDX11 on tumor growth were explored in a xenograft nude mouse model. The relationship between DDX11 and transcription factor Yin Yang-1 (YY1) was researched using the dual luciferase report assay and chromatin immunoprecipitation assay. DDX11 expression was significantly upregulated in OSCC cells. Knockdown of DDX11 inhibited cell proliferation, induced cell cycle arrest, and suppressed PI3K-AKT pathway, while DDX11 overexpression showed opposite effects. The number of apoptotic cells was increased in DDX11 silenced cells. DDX11 upregulation or knockdown accelerated or suppressed tumor growth in vivo, respectively. Moreover, the YY1 bound and activated the DDX11 promoter, resulting in increasing DDX11 expression. Forced expression DDX11 reversed the anticancer effects of YY1 silencing on OSCC cells. DDX11 has tumor-promoting function in OSCC and is transcriptionally regulated by YY1, indicating that DDX11 may serve as a potential target for the OSCC treatment.
研究领域:
肿瘤学
·
分子生物学
·
细胞生物学
·
转化医学
✓ 已验证
DOI
PubMed
高影响因子
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
·
遗传学
·
RNA生物学
·
CRISPR技术
·
病毒学
·
基因编辑
·
功能基因组学
✓ 已验证
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）