RNP免疫沉淀技术在斑马鱼胸苷酸合成酶基因翻译调控研究中的应用

为检测斑马鱼胸苷酸合成酶基因的表达产物TS蛋白在体内是否结合于自身的mRNA,并进一步检测TS蛋白与c-myc mRNA的结合情况。采用免疫沉淀结合得到相应的RNA,即RNP免疫沉淀技术,进一步结合RT-PCR技术分析与蛋白结合的核酸序列,并用Western蛋白印迹分析了TS蛋白。结果显示,人的TS106单克隆抗体可以免疫沉淀斑马鱼的TS蛋白,抗体免疫沉淀的核酸中含有TS mRNA和c-myc mRNA。在斑马鱼中,TS蛋白在体内结合于自身的TS mRNA,并能结合c-myc mRNA,参与基因的表达调控,阐明了TS的翻译调控机制在体内条件下的作用方式,同时也为构建斑马鱼抗肿瘤药理模型提供了理论基础。     

斑马鱼胸苷酸合成酶与自身mRNA的相互作用

斑马鱼(zebrafish,Danio rerio)是生物学领域中公认的研究脊椎类生物的模式生物.胸苷酸合成酶(thymidylate synthase,TS)是DNA从头合成的限速酶,多年来一直作为肿瘤化疗的重要靶酶.前期的研究表明,人和大肠杆菌中TS能与自身的mRNA结合,在翻译水平上具有反馈抑制自调控现象.斑马鱼作为药物模型的研究已成为热点研究领域,为了探讨斑马鱼的胸苷酸合成酶的调控规律,以及与人TS的相关性,利用原核表达,纯化获得高均一性斑马鱼TS蛋白,采用凝胶迁移研究了TS和其mRNA的体外结合,采用免疫共沉淀:RT-PCR技术研究了它们在体内的相互作用,实验结果表明,斑马鱼的TS在体内外均与自身的mRNA存在特异性的相互作用.研究说明,斑马鱼和人的TS具有高度生物学过程相关性,为构建斑马鱼抗肿瘤药理模型提供了理论基础.     

胸苷酸合成酶mRNA亲和多肽的鉴定及其对翻译过程的抑制作用

胸苷酸合成酶(thymidylate synthase, TS)是催化生物体内胸苷酸合成所必需的酶, 多年来一直作为肿瘤化疗的靶点. 研究表明, TS是一种RNA结合蛋白, 可以与其自身mRNA的2个位点结合, 使mRNA翻译受阻. 本文以mRNA体外展示技术进行了由大容量多肽库(>10¹³)中筛选胸苷酸合成酶mRNA亲和多肽的研究, 对随机库进行了12轮循环的选择及扩增. 结果表明, 与初始库相比, 经选择循环之后, 碱性氨基酸及芳香族的苯丙氨酸含量明显增加, 它们在TS RNA与蛋白质的相互作用中扮演着重要角色. 按其理化特性对每一随机位点的氨基酸进行分类, 并与初始库比较, 发现位点1, 12, 17和18具有明显的带正电荷的特性, 表明碱性侧链参与了与RNA的结合. 二级结构预测表明, 随着筛选的进行, 与TS mRNA 亲和的多肽显示出明显的螺旋倾向, 而且形成螺旋的区域富含碱性氨基酸. 凝胶迁移及体外翻译实验证实, 选择循环之后的多肽能够与TS mRNA高度亲和, 并能抑制TS mRNA的翻译. 本研究表明mRNA体外展示方法得到的亲和多肽可以用作新的TS RNA的翻译抑制剂, 并有可能成为一类新型的抗肿瘤药物.     

CSP介导的害虫抗药性新机制研究进展

CSPs(Chemosensory proteins)即化学感受蛋白,其在昆虫体内各个阶段均有表达,参与昆虫的多种生理过程,具有十分复杂的化学功能。CSPs基因介导昆虫抗药性是最新发现的害虫抗药性新机制,且近几年在几种昆虫中被报道。CSPs可以通过螯合作用大量结合农药,进而导致昆虫产生抗药性,但CSPs与杀虫剂的结合机理及其表达调控机制尚未被阐明。基于目前现状,本文系统综述了CSPs在昆虫抗药性中的功能以及抗药性相关酶的表达调控机制等方面的研究进展,分析其表达调控的可能机制,旨在为害虫抗药性机制研究提供新思路。     

害虫抗药性适合度与内分泌调控研究进展

本文就害虫抗药性适合度与内分泌调控研究进展作了概述。害虫抗药性品系与敏感品系之间在形态、生理反应和生物学特性等方面存在差异 ,通常被认为是抗性基因适应性代价的表现 ,并可能由抗性基因引起的抗性害虫体内生理代谢平衡发生变化产生的。目前就抗性代价的形成机制存在 2种假说 :资源限制假说和干扰代谢平衡假说。害虫抗性和敏感品系的生物适合度差异与其体内激素水平及调控能力变化有关。激素对抗药性相关酶系表达调控的可能机制可分为三类 ,即直接调控、间接调控和共同调控     

细菌非编码小RNA分子RyhB的结构与功能

RyhB是一种大小为90个核苷酸的细菌非编码小RNA分子(small noncoding RNA, sRNA).当铁缺乏时,RyhB通过下调一系列与铁的储存和利用相关蛋白的表达水平以维持体内的铁平衡,而其本身的表达则受到负调控因子Fur(ferric uptake regulator)的调节.在体内,RyhB与Hfq蛋白和核糖核酸酶E (ribonuclease E, RNase E)形成核蛋白复合物sRNP来发挥活性.sRNP通过RyhB与靶基因的互补配对序列作用于靶基因的核糖体结合位点,阻断靶mRNA的翻译,并迅速引起靶mRNA的降解.此外,RyhB还可以通过影响致病菌的生物膜形成、趋化性、耐酸性等方面的能力对细菌的致病力进行调节.本文综述了RyhB的结构、功能及作用机制方面的研究进展,并对其存在的生理意义进行了探讨.     

中国淋巴囊肿病毒胸苷酸合酶基因结构特点及分析

胸苷酸合酶(Thymidylate synthase,TS)是进行DNA合成所必需的酶类,与细胞分化及肿瘤发生密切相关。淋巴囊肿病毒属于虹彩病毒科成员,是能引起百余种淡、海水鱼感染,并产生肿瘤的病毒病原。在已完成中国淋巴囊肿病毒株(Lymphocystis disease virus-China,LCDV-C)基因组序列测定的基础上,本文对位于LCDV-C基因组开放阅读框ORF011L的TS基因结构、及其推定蛋白结构进行了分析。该基因全长858bp,GC含量为28．2％,编码一个长为286aa、分子量为32．7kD、等电点为7．1的推定蛋白,称之为中国淋巴囊肿病毒胸苷酸合酶(LCDV-CTS)。该酶所具有的叶酸结合区在第44和70位氨基酸之间,dUMP结合区在第163和206位氨基酸之间,24个必需氨基酸具有高度保守性。二级结构预测结果显示LCDV-C TS含8个a螺旋,6个β折叠和23个环,表明其具备酶活性分子所有的柔性和可变性结构特征。这是迄今所知在脊椎动物虹彩病毒中唯一含两个完整结合区的TS。对来自包括LCDV在内二十个物种的TS结构进行同源性分析,显示LCDV-C TS被单独分为一枝。进一步对LCDV-C TS可能的起源途径及与宿主的作用等进行了探讨。     

中国淋巴囊肿病毒胸苷酸合酶基因结构特点及分析

胸苷酸合酶(Thymidylate synthase,TS)是进行DNA合成所必需的酶类,与细胞分化及肿瘤发生密切相关.淋巴囊肿病毒属于虹彩病毒科成员,是能引起百余种淡、海水鱼感染,并产生肿瘤的病毒病原.在己完成中国淋巴囊肿病毒株(Lymphocystis disease virus-China,LCDV-C)基因组序列测定的基础上,本文对位于LCDV-C基因组开放阅读框ORF 011L的TS基因结构、及其推定蛋白结构进行了分析.该基因全长858bp,GC含量为28.2%,编码一个长为286aa、分子量为32.7kD、等电点为7.1的推定蛋白,称之为中国淋巴囊肿病毒胸苷酸合酶(LCDV-CTS).该酶所具有的叶酸结合区在第44和70位氨基酸之间,dUMP结合区在第163和206位氨基酸之间,24个必需氨基酸具有高度保守性.二级结构预测结果显示LCDV-C TS含8个α螺旋,6个β折叠和23个环,表明其具备酶活性分子所有的柔性和可变性结构特征.这是迄今所知在脊椎动物虹彩病毒中唯一含两个完整结合区的TS.对来自包括LCDV在内二十个物种的TS结构进行同源性分析,显示LCDV-CTS被单独分为一枝.进一步对LCDV-C TS可能的起源途径及与宿主的作用等进行了探讨.     

植物miRNA的分子特征及其在逆境中的响应机制

逆境胁迫是影响植物生长发育、生物产量与品质形成的主要因素之一。通过诱导表达抗逆有关的编码基因与部分非编码基因是植物响应逆境的主要方式。miRNA作为一种非编码基因在植物生长、发育以及抗逆等过程中起重要的调控作用。研究表明:逆境胁迫下miRNA可以形成miRNA诱导沉默复合物(miRNA-induced silencing complex,miRISC),并与靶mRNA互补配对结合,进而引起靶mRNA的降解或者抑制其翻译,从而实现对下游抗逆相关基因表达的调控,最终引起代谢与信号转导途径的变化实现对逆境的响应。本文从植物逆境胁迫下诱导miRNA的产生、靶基因的识别以及作用机制等方面进行了综述。     

CPR和P450基因在昆虫抗药性中的作用研究进展

P450酶系在昆虫代谢农药中有重要作用,NADPH-细胞色素P450还原酶（NADPH-cytochrome P450 reductase,CPR）和细胞色素P450（P450）在该酶系起核心作用。昆虫具有P450超基因家族,但只有一个单一的CPR基因,CPR是昆虫所有参与农药代谢的P450酶的唯一电子供体,其影响P450活性。P450基因的高水平表达在害虫抗药性中具有重要作用,P450基因介导的昆虫抗药性是最重要的代谢抗性类型。不同P450基因的高表达的调控机制不同,引起P450基因过量表达的原因可能有P450基因的编码区突变、顺式作用元件和反式作用因子变化、基因扩增等。细胞色素P450介导的抗药性存在一定程度的进化可塑性,即同种昆虫不同种群对相同的农药产生抗药性时,导致抗性产生的P450基因不同;同一昆虫品系在某种农药的抗性选择压力下,影响抗性的P450基因的种类和表达特性会随着持续的农药选择而发生变化。最近的研究显示,CPR的变异和昆虫抗药性相关,但是昆虫CPR基因介导抗药性的机制还缺乏深入研究。全面阐释P450酶系介导昆虫抗药性的机制、建立基于P450基因表达量变化与CPR突变的抗性分子标记,对于害虫抗药性治理具有重要意义。     

Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.     

Effect of 2'-O-methyl antisense ORNs on expression of thymidylate synthase in human colon cancer RKO cells

Translation of thymidylate synthase (TS) mRNA is controlled by its own protein end-product TS in a negative autoregulatory manner. Disruption of this regulation results in increased synthesis of TS and may lead to the development of cellular drug resistance to TS-directed anticancer agents. As a strategy to inhibit TS expression, antisense 2′-O-methyl RNA oligoribonucleotides (ORNs) were designed to directly target the 5′ upstream cis-acting regulatory element (nucleotides 80–109) of TS mRNA. A 30 nt ORN, HYB0432, inhibited TS expression in human colon cancer RKO cells in a dose-dependent manner but had no effect on the expression of β-actin, α-tubulin or topoisomerase I. TS expression was unaffected by treatment with control sense or mismatched ORNs. HYB0504, an 18 nt ORN targeting the same core sequence, also repressed expression of TS protein. However, further reduction in oligo size resulted in loss of antisense activity. Following HYB0432 treatment, TS protein levels were reduced by 60% within 6 h and were maximally reduced by 24 h. Expression of p53 protein was inversely related to that of TS, suggesting that p53 expression may be directly linked to intracellular levels of TS. Northern blot analysis demonstrated that TS mRNA was unaffected by HYB0432 treatment. The half-life of TS protein was unchanged after antisense treatment suggesting that the mechanism of action of antisense ORNs is mediated through a process of translational arrest. These findings demonstrate that an antisense ORN targeted at a critical cis-acting element on TS mRNA can specifically inhibit expression of TS protein in RKO cells.     

The identification of thymidylate synthase peptide domains located in the interface region that bind thymidylate synthase mRNA

Thymidylate synthase (TS) is a critical chemotherapeutic target and intracellular levels of TS are an important determinant of sensitivity to TS inhibitors. Translational autoregulation represents one cellular mechanism for controlling the level of expression of TS. This mechanism involves the binding of TS protein to its own messenger RNA (mRNA), thus, repressing translational efficiency. The presence of excess substrate or inhibitors of TS leads to derepression of protein binding to mRNA, resulting in increased translational efficiency and ultimately increased levels of TS protein. TS protein has been shown to bind to two distinct areas on its mRNA. The goal of the present work is to define the TS domains responsible for this interaction. Using a separate series of overlapping 17-mer peptides spanning the length of both the human and Escherichia coli TS sequences, we have identified six potential domains located in the interface region of the TS protein that bind TS mRNA. The identified domains that bind TS mRNA include three concordant regions in both the human and E. coli peptide series. Five of the six binding peptides contain at least one invariant arginine residue, which has been shown to be critical in other well-defined protein-RNA interactions. These data suggest that the identified highly conserved protein domains, which occur at the homodimeric interface of TS, represent potential participating sites for binding of TS protein to its mRNA.     

Role of cysteine amino acid residues on the RNA binding activity of human thymidylate synthase

The role of cysteine sulfhydryl residues on the RNA binding activity of human thymidylate synthase (TS) was investigated by mutating each cysteine residue on human TS to a corresponding alanine residue. Enzymatic activities of TS:C43A and TS:C210A mutant proteins were nearly identical to wild-type TS, while TS:C180A and TS:C199A mutants expressed >80% of wild-type enzyme activity. In contrast, TS:C195A was completely inactive. Mutant proteins, TS:C195A, TS:C199A and TS:C210A, retained RNA binding activity to nearly the same degree as wild-type human TS. RNA binding activity of TS:C43A was reduced by 30% when compared to wild-type TS, while TS:C180A was completely devoid of RNA binding activity. In vitro translation studies confirmed that mutant proteins TS:C43A, TS:C195A, TS:C199A and TS:C210A, significantly repressed human TS mRNA translation, while TS:C180A was unable to do so. To confirm the in vivo significance of the cysteine sulfhydryl residue, mutant proteins TS:C180A and TS:C195A were each expressed in human colon cancer HCT-C18:TS(–) cells that expressed a functionally inactive TS. A recombinant luciferase reporter gene under the control of a TS-response element was co-transfected into these same cells, and luciferase activity increased in the presence of the TS:C195A mutant TS protein to a level similar to that observed upon expression of wild-type TS protein. In contrast, luciferase activity remained unchanged in cells expressing the TS:C180A mutant protein. Taken together, these findings identify Cys-180 as a critical residue for the in vitro and in vivo translational regulatory effects of human TS.     

Characterization of a cis-acting regulatory element in the protein coding region of thymidylate synthase mRNA

Thymidylate synthase (TS) functions as an RNA-binding protein by interacting with two different sequences on its own mRNA. One site is located in the 5′-upstream region of human TS mRNA while the second site is located within the protein coding region corresponding to nt 434–634. In this paper, a 70 nt RNA sequence, corresponding to nt 480–550, was identified that binds TS protein with an affinity similar to that of full-length TS mRNA and TS434–634 RNA. In vitro translation studies confirmed that this sequence is critical for the translational autoregulatory effects of TS. To document in vivo biological significance, TS sequences contained within this region were cloned onto the 5′-end of a luciferase reporter plasmid and transient transfection experiments were performed using H630 human colon cancer cells. In cells transfected with p644/TS434–634 or p644/TS480–550, luciferase activity was decreased 2.5-fold when compared to cells transfected with p644 plasmid alone. Luciferase mRNA levels were identical for each of these conditions as determined by RNase protection and RT–PCR analysis. Immunoprecipitation of TS ribonucleoprotein complexes revealed a direct interaction between TS protein and TS480–550 RNA in transfected H630 cells. Treatment with 5-fluorouridine resulted in a nearly 2-fold increase in luciferase activity only in cells transfected with p644/TS434–634 and p644/TS480–550. This study identifies a 70 nt TS response element in the protein coding region of TS mRNA with in vitro and in vivo translational regulatory activity.