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Efficient genome editing in Claviceps purpurea using a CRISPR/Cas9 ribonucleoprotein method

Synthetic and Systems Biotechnology 2022年第2期7卷 664-670页

作者： Lu Yu Meili Xiao Zhihua Zhu Yinmei Wang Zhihua Zhou Pingping Wang Gen Zou CAS-Key Laboratory of Synthetic Biology CAS Center for Excellence in Molecular Plant SciencesInstitute of Plant Physiology and EcologyChinese Academy of Sciences300 Fenglin RdShanghai200032China University of Chinese Academy of Sciences Beijing100049China Shanghai Key Laboratory of Agricultural Genetics and Breeding Institute of Edible FungiShanghai Academy of Agriculture Science1000 Jinqi RdFengxianShanghai201403China

Claviceps purpurea produces many pharmacologically important ergot alkaloids(EAS),which are widely used to treat migraine and hypertension and to aid childbirth.Although an EAS biosynthetic cluster of C.purpurea has been discovered more than 20 years ago,the complete biosynthetic pathway of EAS has not been fully characterized until now.The main obstacle to elucidating this pathway and strain modification is the lack of efficient genome-editing tools for C.purpurea.The conventional gene manipulation method for C.purpurea relies on homologous recombination(HR),although the efficiency of HR in C.purpurea is very low(~1-5%).Consequently,the disruption of target genes is laborious and time-consuming.Although CRISPR/Cas9 genome-editing methods based on in vivo Cas9 expression and gRNA transcription have been reported recently,their gene-disruption efficiency is still very low.Here,we developed an efficient genome-editing system in C.purpurea based on in vitro assembled CRISPR/Cas9 gRNA ribonucleoprotein complexes.As proof of principle,three target genes were efficiently knocked out using this CRISPR/Cas9 ribonucleoprotein complex-mediated HR system,with editing efficiencies ranging from 50%to 100%.Inactivation of the three genes,which are closely related to uridine biosynthesis(ura5),hypha morphology(rac),and EAS production(easA),resulted in a uridine auxotrophic mutant,a mutant with a drastically different phenotype in axenic culture,and a mutant that did not produce EAS,respectively.Our ribonucleoprotein-based genome-editing system has a great advantage over conventional and in vivo CRISPR/Cas9 methods for genome editing in C.purpurea,which will greatly facilitate elucidation of the EAS biosynthetic pathway and other future basic and applied research on C.purpurea.

关键词： ribonucleoprotein Genome editing Ergot alkaloids Biosynthetic pathway Homologous recombination

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Dengue virus non-structural 1 protein interacts with heterogeneous nuclear ribonucleoprotein H in human monocytic cells

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Asian Pacific Journal of Tropical Medicine 2016年第2期9卷 109-114页

作者： Drishya Diwaker Mishra K P Ganju L Singh S B Immunomodulation Lab Defence Institute of Physiology & Allied Sciences

Objective: To study protein-protein interaction between heterogeneous nuclear ribonucleoprotein H(hn RNP H) and Dengue virus(DENV) proteins. Methods: DENV proteins were screened against the host hn RNP H protein, in order to identify the host-viral protein-protein interactions in DENV infected THP-1 cells by co-immunoprecipitation. The co-localization of the interacting proteins was further confirmed by immunofluorescence microscopy. Results: The host protein hn RNP H was found to interact with DENV nonstructural 1 protein and help the virus to multiply in the cell. Conclusions: The non-structural 1 glycoprotein is a key modulator of host immune response and is also involved in viral replication. Therefore, disruption of this key interaction between hn RNP H and DENV nonstructural 1 could be an important therapeutic strategy for management of DENV infection.

关键词： Dengue virus Non-structural 1 protein Heterogeneous nuclear ribonucleoprotein H Protein interactions

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Enhanced Bombyx genome editing via Cas9 ribonucleoprotein injection

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Insect Science 2019年第6期26卷 1059-1062页

作者： Ling Jia San-Yuan Ma Tong Zhang Wei-Qing Xing Yue Liu Yu-Feng Li Xiao-Xu Chen Qing-You Xia State Key Laboratory of Silkworm Genome Biology Southwest University Chongqing China Chongqing Engineering and Technology Research Center for Novel Silk Materials Southwest University Chongqing China

Dear Editor, The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has become a popular and powerful method for manipulating genomes with considerably little effort and high efficiency. It allows for the generation of targeted indels, genomic structure variations, and insertion of foreign fragments. However, increasing concerns have been raised about possible off-target effects.

关键词： Bombyx genome ribonucleoprotein increasing concerns

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Protein and gene expression characteristics of heterogeneous nuclear ribonucleoprotein H1 in esophageal squamous cell carcinoma

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World Journal of Gastroenterology 2016年第32期22卷 7322-7331页

作者： Yu-Lin Sun Fei Liu Fang Liu Xiao-Hang Zhao State Key Laboratory of Molecular Oncology National Cancer Center/Cancer Hospital Chinese Academy of Medical Sciences and Peking Union Medical College

AIM To investigate the expression characteristics of heterogeneous nuclear ribonucleoprotein H1(HNRNPH1) m RNA and protein in cell lines and tissues of esophageal squamous cell carcinoma(ESCC). METHODS Western blotting was used to assess the expression of HNRNPH1 protein in seven ESCC cell lines and 30 paired fresh tissue specimens. The subcellular localization of HNRNPH1 was determined by immunofluorescence in ESCC cells. The RNA sequencing data from 87 patients with ESCC were obtained from the cancer genome atlas(TCGA), and the expression and clinical characteristics analysis of different transcript variants of HNRNPH1 were evaluated in this dataset. In addition, immunohistochemistry was carried out to detect the expression of HNRNPH1 protein in 125 patients.RESULTS The expression of HNRNPH1 protein varied across different ESCC cell lines. It was exclusively restricted to the nucleus of the ESCC cells. There are two transcript variants of the HNRNPH1 gene. Variant 1 was constitutively expressed, and its expression did not change during tumorigenesis. In contrast, levels of variant 2 were low in non-tumorous tissues and were dramatically increased in ESCC(P = 0.0026). The high levels of variant 2 were associated with poorer differentiated tumors(P = 0.0287). Furthermore, in paired fresh tissue specimens, HNRNPH1 protein was overexpressed in 73.3%(22/30) of neoplastic tissues. HNRNPH1 was significantly upregulated in ESCC, with strong staining in 43.2%(54/125) of tumor tissues and 22.4%(28/125) of matched non-cancerous tissues(P = 0.0005). Positive HNRNPH1 expression was significantly associated with poor tumor differentiation degree(P = 0.0337).CONCLUSION The different alternative transcript variants of HNRNPH1 exhibited different expression changes during tumorigenesis. Its m RNA and protein were overexpressed in ESCC and associated with poorer differentiation of tumor cells. These findings highlight the potential of HNRNPH1 in the therapy and diagnosis of ESCC.

关键词： Heterogeneous nuclear ribonucleoprotein H1 Esophageal squamous cell carcinoma Alternative transcript variants Biomarker

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Gene Expression Changes Associated with the Loss of Heterogeneous Nuclear ribonucleoprotein M Function

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American Journal of Molecular Biology 2017年第2期7卷 87-98页

作者： Jun-ichi Takino Kentaro Nagamine Mikoto Suzuki Akiko Sakasai-Sakai Masayoshi Takeuchi Takamitsu Hori Department of Biochemistry Hiroshima International University Hiroshima Japan Department of Advanced Medicine Kanazawa Medical University Ishikawa Japan

Advanced glycation endproducts (AGEs) are formed by the nonenzymatic reaction of sugars with proteins. Glycation may adversely affect proteins, such as by inducing a loss of function. It has been shown that glyceraldehyde-derived AGEs (Glycer-AGEs) accumulate in the liver of patients with nonalcoholic steatohepatitis (NASH). Previously, we showed the formation of intracellular Glycer-AGEs upon exposure of hepatocytes to fructose in vitro, and identified an RNA-binding protein, heterogeneous nuclear ribonucleoprotein M (HNRNPM), as a target for glycation. However, the impact of glycated HNRNPM in NASH remains poorly understood. In this study, we examined gene expression changes caused by HNRNPM knockdown, and investigated the up- and down-regulated genes as noninvasive biomarker candidates for NASH. Microarray analysis after HNRNPM knockdown showed that the levels of 138 transcripts were increased, while those of 100 transcripts were decreased as compared with those in the control. Gene Ontology-based functional analysis showed that 14 upregulated and 9 downregulated genes were associated with the extracellular space, which may enable their detection using blood tests. Among these, six of the up- and down-regulated genes were associated with the extracellular exosome. These results suggest that the loss of HNRNPM function by glycation is reflected extracellularly. Therefore, the identified genes may serve as noninvasive biomarkers for Glycer-AGEs-related NASH.

关键词： Glycation Nonalcoholic Steatohepatitis Heterogeneous Nuclear ribonucleoprotein M Biomarkers

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Cloning and characterization of a novel deletion mutant of heterogeneous nuclear ribonucleoprotein M4 from human dendritic cells

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Science China(Life Sciences) 2000年第6期43卷 648-654页

作者：黄欣赵忠良袁正隆张明徽朱学军陈国友曹雪涛 Department of Immunology Second Military Medical University Shanghai 200433 China

To identify differentially expressed genes from antigen-stimulated human dendritic cells (DC), subtractive cloning was adopted and more than ten novel genes differentially expressed were cloned. One is a deletion mutant of heterogeneous nuclear ribonucleoprotein (hnRNP) M4 in which the residues from 159 to 197 of hnRNP M4 have been absent. The deletion mutant was shown to- be co-expressed with hnRNP M4 in cell lines. The mutant was expressed in antigen-stimulated DC but not in normal DC. Northern blot analysis revealed the presence of a major hnRNP M4 deletion mutant mRNA transcript of 2.4 kilobase with the highest levels in peripheral lymphocytes, lung, liver and spleen. It was also expressed in bone marrow-derived stromal cells (BMSC), BMSC treated with several cytokines but not in BMSC treated with TNF-a. The results revealed a new member of hnRNP family and suggested that hnRNP would participate in antigen process and presentation.

关键词： dendritic cells subtractive hybridization gene cloning heterogeneous nuclear ribonucleoprotein deletion mutant.

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Insight into the mechanisms and functions of spliceosomal snRNA pseudouridylation

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World Journal of Biological Chemistry 2014年第4期5卷 398-408页

作者： Hironori Adachi Yi-Tao Yu Department of Biochemistry and Biophysics Center for RNA Biology University of Rochester Medical Center Rochester NY 14642 United States

Pseudouridines(Ψs) are the most abundant and highly conserved modified nucleotides found in various stable RNAs of all organisms. Most Ψs are clustered in regions that are functionally important for pre-m RNA splicing. Ψ has an extra hydrogen bond donor that endows RNA molecules with distinct properties that contribute significantly to RNA-mediated cellular processes. Experimental data indicate that spliceosomal sn RNA pseudouridylation can be catalyzed by both RNA-dependent and RNA-independent mechanisms. Recent work has also demonstrated that pseudouridylation can be induced at novel positions under stress conditions, suggesting a regulatory role for Ψ.

关键词： Pre-m RNA splicing U2 sn RNA Box H/ACA ribonucleoprotein Pseudouridine Induced RNA modification

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The RFA regulatory sequence-binding protein in the promoter of prostate-specific antigen gene

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Science China(Life Sciences) 2003年第2期46卷 184-193页

作者：陈蔚文张建业 Charles Y F Young 张莲英陈留存赵健 Institute of Biochemistry and Molecular Biology School of Medicine Shandong University Jinan 250012 China Department of Urology Research of Mayo Clinic/Foundation MN 55905USA Department of Thoracic Surgery Qilu Hospital of Shandong University Jinan 250012 China

To assure what sequence associated with the androgen regulation, a 15 bp region at the upstream of the ARE of prostate-specific antigen (PSA) promoter, termed RFA, was found in-dispensable for androgen receptor (AR)-mediated transactivation of PSA promoter. In transfection and CAT assays, some nucleotides substitution in RFA could significantly decrease the androgen inducibility for PSA promoter. The in vitro DNA binding assay demonstrated that RFA bound spe-cifically with some non-receptor protein factors in prostate cell nucleus, but the mutant type of RFA lost this ability, so RFA might be a novel accessory cis-element. The RFA-binding proteins were isolated and purified by affinity chromatography using RFA probes. SDS-PAGE and preliminary protein identification showed these proteins possessed sequence high homology with multifunc-tional protein heterogeneous nuclear ribonucleoprotein A1, A2 (hnRNP A1, A2). RFA-binding pro-teins possibly cooperate with AR-mediated transactivation for PSA promoter as coactivator. The study results will facilitate further understanding the mechanism and tissue specificity of PSA pro-moter.

关键词： prostate-specific antigen, promoter, heterogeneous nuclear ribonucleoprotein A1, A2.

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细胞体内分离鉴定RNA结合蛋白结合的靶mRNA技术的建立和验证

细胞体内分离鉴定RNA结合蛋白结合的靶mRNA技术的建立和验证

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作者：狄文娜中国协和医科大学

学位级别：硕士

转录后水平的基因表达调控是细胞生长和分化过程中最重要的调节机制之一。转录后水平的调控会影响很多基因的蛋白表达水平和细胞内蛋白表达的特定位置，从而影响细胞的分化方向。RNA结合蛋白是转录后水平基因表达调控的主要承担者。RNA... 详细信息

转录后水平的基因表达调控是细胞生长和分化过程中最重要的调节机制之一。转录后水平的调控会影响很多基因的蛋白表达水平和细胞内蛋白表达的特定位置，从而影响细胞的分化方向。RNA结合蛋白是转录后水平基因表达调控的主要承担者。RNA结合蛋白(RNA—binding protein，RBP)多具有保守的RNA结合模体(RNA—binding motifs，RBM)，其所结合的靶RNA也多具有特异结构或序列，RNA结合蛋白通过RNA结合模体来特异地与靶RNA的特异结构或序列结合，进而调节这些RNA的剪辑、运输、稳定性和翻译等。体内分离和鉴定RNA结合蛋白的核糖核蛋白复合体已成为基因组后时期的一个具有重要意义的挑战。本工作建立了一种能够全景式分离鉴定RNA结合蛋白靶mRNA的技术平台。本技术平台的路线是：分别构建生物素连接酶(BirA)基因、可生物素化多肽标签(BAP)—RNA结合蛋白融合基因的真核表达载体。共转染上述真核表达载体至细胞内使其表达，在生物素存在的前提下，利用生物素连接酶(BirA酶)使可被生物素化的多肽标签进行生物素化，通过Streptavidin Sepharose—beads的一步纯化，即可获得与多肽标签融合表达的RNA结合蛋白及其靶mRNA，即核糖核蛋白复合体ribonucleoprotein，RNP。工作中通过Western blot方法验证了该项技术路线的可靠性，表明Streptavidin Sepharose—beads的一步纯化法可以简便得到核糖核蛋白复合体。本工作还通过RT-PCR方法检测已知PCBP2蛋白所结合的靶mRNA：Trx2、Cln2、PPT2和coxⅡ mRNA。

关键词： BirA BAP RNA结合蛋白 PCBP2 ribonucleoprotein 靶mRNA

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Regulation of RNA binding proteins in trypanosomatid protozoan parasites

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World Journal of Biological Chemistry 2016年第1期7卷 146-157页

作者： María Albertina Romaniuk Gabriela Cervini Alejandro Cassola Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús UNSAM-CONICET

Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins(RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3' untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of m RNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy.

关键词： Trypanosoma Posttranscriptional gene expression ribonucleoprotein complexes RNA-binding protein Developmental regulation Sleeping sickness Posttranslational modification Phosphorylation Chagas disease

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