核糖体蛋白基因表达与肿瘤的关系

核糖体蛋白是组成核糖体的主要成分,在细胞内蛋白质生物合成中发挥重要作用。近来人们发现,核糖体具有参与DNA修复、细胞发育调控和细胞分化等核糖体外功能。并且在胃癌、结直肠癌、食管癌和肝癌等肿瘤组织中一些核糖体蛋白基因高表达,通过对肿瘤组织中核糖体蛋白基因高表达的深入研究,可以进一步阐明肿瘤发生、发展的机制,了解核糖体蛋白基因高表达在恶性肿瘤中的作用,为肿瘤的基因诊断和基因治疗开辟一个新的研究领域。     

水稻25S核糖体RNA2′—O—核糖甲基化位点的测定

核糖 2′ O 甲基化修饰是真核生物核糖体RNA上的一种极为普遍的修饰方式。为了测定水稻 2 5S核糖体RNA上发生甲基化修饰的具体位点 ,设计并纯化了一系列与水稻 2 5S和酵母 2 8S核糖体RNA均配对的引物 ,在测定水稻核糖体RNA甲基化位点的同时 ,将酵母核糖体RNA甲基化位点的测定作为对照 ,在同一条件下 ,分别以水稻及酵母总RNA为模板进行dNTP浓度依赖的引物延伸反应。在测得的水稻甲基化位点中 ,有 3 1个位点是与酵母共有的 ,占酵母 2 8S核糖体RNA的甲基化位点总数的 80 %以上。另外 ,通过与已经测定的拟南芥 2 5S核糖体RNA上的甲基化位点进行比较 ,在水稻中又确定了与拟南芥相同的 5 4个甲基化位点。最终在水稻 2 5S核糖体RNA中 ,初步确定了 85个甲基化位点 ,并绘制了水稻 2 5S核糖体RNA的甲基化位点分布图。这些结果表明在不同的真核生物中 ,核糖体RNA上大部分位点核糖的甲基化修饰是保守的 ,而且亲缘关系越近 ,其保守性越强。结果还表明 ,高等植物核糖体RNA上有大量的核糖甲基化修饰位点 ,并且其中相邻的位点均被甲基化修饰的数量明显高于其他生物。所测得的甲基化位点将为进一步寻找植物中新的C/D框小分子核仁RNA(sonRNA)提供重要的依据     

核糖体蛋白S26的研究进展

核糖体蛋白(ribosomal proteins,RPs)不仅在细胞内参与合成蛋白质,还具有多种核糖体外功能。核糖体蛋白S26(RPS26)位于核糖体小亚基,其功能障碍与多种疾病密切相关。近年来,有关RPS26的研究主要在参与核糖体装配等核糖体功能方面,以及参与无义介导的mRNA降解机制(nonsense-mediated mRNA decay,NMD)、直接或间接调控重要的抑癌基因p53表达等核糖体外功能方面。多篇报道证实RPS26基因突变可引起戴-布二氏贫血(Diamond-Blackfan anemia,DBA),而RPS26基因与Ⅰ型糖尿病的关系仍有争议。探索RPS26参与NMD机制在DBA发生中的作用有助于深入认识DBA发病机理,同时也可为完善SMaRT(spliceosome-mediated mRNA trans-splicing)技术等基因疗法提供帮助。此外,RPS26在癌症中的作用也值得进一步探索。     

SUMO化修饰的Apak对核糖体RNA合成的调控作用

核糖体是由核糖体RNA和核糖体蛋白组成的复合体,其功能是参与蛋白质合成.SUMO化修饰的底物蛋白对核糖体的形成有重要调控作用.前期研究发现,KRAB型锌指蛋白Apak能特异地抑制p53所介导的凋亡通路.进一步研究发现,在核仁应激及癌基因激活条件下,抑癌蛋白ARF促进Apak发生SUMO化修饰并促使其移位于核仁.为了进一步探讨SUMO化修饰的Apak对核糖体RNA合成的调控功能,本研究通过Northern blot检测SUMO化修饰的Apak对核糖体RNA合成的影响,实时定量PCR检测核糖体RNA转录水平,RNA-Ch IP方法检测核糖体RNA与Apak蛋白的相互作用,结果表明,SUMO化修饰的Apak抑制47S核糖体RNA前体的合成且抑制RNA聚合酶Ⅰ介导转录的18S和5.8S r RNA的合成;在放线菌素D以及癌基因诱导下,促进Apak与18S,5.8S r RNA相互作用.本研究对理解Apak的功能和作用机制提供了新的依据,为深入研究KRAB型锌指蛋白家族分子对核糖体RNA的调控奠定了基础.     

从基因组分析贾第虫的核糖体合成系统

为探讨贾第虫细胞核内核糖体合成系统,及与典型的真核生物有何差异,首先,确定在典型真核生物中参与核糖体合成的129条共有的保守蛋白,然后用这些蛋白搜索贾第虫基因组以调查它们在贾第虫中的直系同源蛋白的情况,以对贾第虫的核糖体合成系统作一了解。结果表明：贾第虫具有89条这些蛋白的直系同源蛋白,包括参与rRNA甲基化和假尿嘧啶化的蛋白复合体成员,以及存在于90S、40S和60S复合体中的蛋白。贾第虫的核糖体合成系统与典型的真核生物相似,但还有40条蛋白在贾第虫基因组中找不到同源蛋白。这意味着贾第虫的核糖体合成系统较典型的真核生物简单。贾第虫虽然没有核仁结构,但其核糖体亚基合成的途径和机制可能与真核细胞相似,参与的成分不同于无核仁结构的原核生物,可能相对简单。     

泛肽、核糖体蛋白及泛肽-核糖体蛋白S27a与肿瘤的关系

泛肽-核糖体蛋白S27a(Ubiquitin-ribosomal protein S27a,UBRS27a)是泛肽和核糖体蛋白的融合蛋白,N端为泛肽,C端由含C2-C2型锌指结构域的高度保守核糖体蛋白S27a构成。在真核细胞中表达时,被酶解成泛肽和核糖体蛋白。该多功能核糖体蛋白在各种活性增殖细胞和瘤组织中高度表达,在多种类型的肿瘤细胞中,该基因的过量表达是一个典型特征。本实验室对该蛋白在家蚕中的作了初步研究,也发现RPS27a在活性增殖细胞中表达量很高。大多数核糖体蛋白的功能还没有完全探明,它们不仅仅在组装成核糖体时起作用,往往还有核糖体外的功能。回顾了最近几年有关该融合蛋白以及与它相关的泛肽途径、核糖体蛋白与肿瘤之间的关系。通过对它们的研究,有可能预示肿瘤的发生和发展,并为肿瘤临床诊断提供依据,为恶性肿瘤的治疗提供靶点。     

泛肽、核糖体蛋白及泛肽-核糖体蛋白S27a与肿瘤的关系

泛肽-核糖体蛋白S27a（Ubiquitin-ribosomal protein S27a,UBRS27a）是泛肽和核糖体蛋白的融合蛋白,N端为泛肽,C端由含C2-C2型锌指结构域的高度保守核糖体蛋白S27a构成。在真核细胞中表达时,被酶解成泛肽和核糖体蛋白。该多功能核糖体蛋白在各种活性增殖细胞和瘤组织中高度表达,在多种类型的肿瘤细胞中,该基因的过量表达是一个典型特征。本实验室对该蛋白在家蚕中的作了初步研究,也发现RPS27a在活性增殖细胞中表达量很高。大多数核糖体蛋白的功能还没有完全探明,它们不仅仅在组装成核糖体时起作用,往往还有核糖体外的功能。回顾了最近几年有关该融合蛋白以及与它相关的泛肽途径、核糖体蛋白与肿瘤之间的关系。通过对它们的研究,有可能预示肿瘤的发生和发展,并为肿瘤临床诊断提供依据,为恶性肿瘤的治疗提供靶点。     

沙葱萤叶甲核糖体蛋白基因GdRpS3a的克隆、分子特性及表达分析

核糖体蛋白(ribosomal protein,Rp)是一类参与蛋白质合成、细胞代谢、机体免疫及信号传导等重要功能的蛋白质.本研究旨在克隆沙葱萤叶甲Galeruca daurica核糖体蛋白S3a基因cDNA全长序列,分析其分子特性和表达模式,以期为进一步研究其在沙葱萤叶甲生长发育及滞育中的作用提供必要的基础.根据现有...     

细胞周期决定核糖体蛋白S6在真核细胞核仁中的聚积

核糖体蛋白S6（rpS6）是核糖体40S小亚基的核心组成蛋白之一。研究表明,rpS6可以通过核定位信号进入细胞核中,在核仁中参与核糖体的组装。在该研究中发现,rpS6在高等真核细胞核仁中的聚积与细胞周期有关,rpS6在S期中晚期开始在核仁中聚积,G2期含量达到最高,M期核仁分解时消失。推测,rpS6在核仁中的这种分布特性可能与核糖体的合成随细胞周期变化有关。     

核糖体S6蛋白激酶的生物学特性和功能

核糖体S6蛋白激酶（ribosomal S6 kinase,RSK）是细胞信号传导通路中的重要成员。1985年Erikson和Mailer在非洲爪蟾卵中发现一种90kD的蛋白激酶,它可以使40S核糖体亚单位S6蛋白发生磷酸化,从而促进某些mRNA的翻译,在调节细胞生长和增殖过程中起重要作用。这种蛋白激酶被命名为RSK或p90rsk。后来发现该蛋白是丝裂原激活蛋白激酶（mitogen—activated protein kinases,MAPKs）的下游底物,可以被MAPKs磷酸化激活,因此,又称为MAPKAP—K1（mitogen—activated protein kinase—activated protein kinase-1）。迄今为止,人们已经发现了4种RSK亚型,它们在高等真核细胞中广泛表达。随着研究的逐步深入,人们发现RSK在多种生命活动中发挥重要作用,包括调节基因转录、参与细胞周期调控、促进细胞增殖和分化、调节细胞生存和凋亡以及参与学习和记忆的形成等。本将简要介绍RSK的结构、激活机制、信号传导通路,以及对细胞功能的调节。     

Composition and functional characterization of yeast 66S ribosome assembly intermediates

The pathway and complete collection of factors that orchestrate ribosome assembly are not clear. To address these problems, we affinity purified yeast preribosomal particles containing the nucleolar protein Nop7p and developed means to separate their components. Nop7p is associated primarily with 66S preribosomes containing either 27SB or 25.5S plus 7S pre-rRNAs. Copurifying proteins identified by mass spectrometry include ribosomal proteins, nonribosomal proteins previously implicated in 60S ribosome biogenesis, and proteins not known to be involved in ribosome production. Analysis of strains mutant for eight of these proteins not previously implicated in ribosome biogenesis showed that they do participate in this pathway. These results demonstrate that proteomic approaches in concert with genetic tools provide powerful means to purify and characterize ribosome assembly intermediates.     

Composition and structure of the 80S ribosome from the green alga Chlamydomonas reinhardtii: 80S ribosomes are conserved in plants and animals

We have conducted a proteomic analysis of the 80S cytosolic ribosome from the eukaryotic green alga Chlamydomonas reinhardtii, and accompany this with a cryo-electron microscopy structure of the ribosome. Proteins homologous to all but one rat 40S subunit protein, including a homolog of RACK1, and all but three rat 60S subunit proteins were identified as components of the C. reinhardtii ribosome. Expressed Sequence Tag (EST) evidence and annotation of the completed C. reinhardtii genome identified genes for each of the four proteins not identified by proteomic analysis, showing that algae potentially have a complete set of orthologs to mammalian 80S ribosomal proteins. Presented at 25A, the algal 80S ribosome is very similar in structure to the yeast 80S ribosome, with only minor distinguishable differences. These data show that, although separated by billions of years of evolution, cytosolic ribosomes from photosynthetic organisms are highly conserved with their yeast and animal counterparts.     

A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export

The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.     

On the role of cyclic AMP-independent protein kinases in the modification of yeast ribosomal proteins in vivo

Two proteins of yeast 40S ribosome subunit and four proteins of the 60S ribosome subunit were labelled in vivo with [32P]orthophosphate. Five of these proteins were phosphorylated by protein kinase 3, an enzyme which is cyclic AMP-independent and uses ATP and GTP as phosphoryl donors. Two proteins, belonging to the 60S ribosome subunit were phosphorylated by another, highly specific, cyclic AMP-independent protein kinase 1 B. Both in vivo and in vitro the most extensively phosphorylated protein species were acidic proteins, L44, L45 (according to the nomenclature of Kruiswijk & Planta, Molec. Biol. Rep., 1, 409-415, 1974) possibly corresponding to bacterial L7 and L12 proteins. The 40S ribosomal protein, S9, analogous to mammalian S6 protein, was phosphorylated in vivo but was not phosphorylated in vitro by either of the cyclic AMP-independent protein kinases. The obtained results clearly indicate that cyclic AMP-independent yeast protein kinases might be involved in the modification in vivo of some ribosomal proteins, in particular of the strongly acidic proteins of 60S ribosome subunit.     

The carboxy-terminal extension of yeast ribosomal protein S14 is necessary for maturation of 43S preribosomes

Eukaryotic ribosomal proteins are required for production of stable ribosome assembly intermediates and mature ribosomes, but more specific roles for these proteins in biogenesis of ribosomes are not known. Here we demonstrate a particular function for yeast ribosomal protein rpS14 in late steps of 40S ribosomal subunit maturation and pre-rRNA processing. Extraordinary amounts of 43S preribosomes containing 20S pre-rRNA accumulate in the cytoplasm of certain rps14 mutants. These mutations not only reveal a more precise function for rpS14 in ribosome biogenesis but also uncover a role in ribosome assembly for the extended tails found in many ribosomal proteins. These studies are one of the first to relate the structure of eukaryotic ribosomes to their assembly pathway-the carboxy-terminal extension of rpS14 is located in the 40S subunit near the 3' end of 18S rRNA, consistent with a role for rpS14 in 3' end processing of 20S pre-rRNA.