RNA recognition and cleavage by a splicing endonuclease

The RNA splicing endonuclease cleaves two phosphodiester bonds within folded precursor RNAs during intron removal, producing the functional RNAs required for protein synthesis. Here we describe at a resolution of 2.85 angstroms the structure of a splicing endonuclease from Archaeglobus fulgidus bound with a bulge-helix-bulge RNA containing a noncleaved and a cleaved splice site. The endonuclease dimer cooperatively recognized a flipped-out bulge base and stabilizes sharply bent bulge backbones that are poised for an in-line RNA cleavage reaction. Cooperativity arises because an arginine pair from one catalytic domain sandwiches a nucleobase within the bulge cleaved by the other catalytic domain.     

Group II introns designed to insert into therapeutically relevant DNA target sites in human cells

Mobile group II intron RNAs insert directly into DNA target sites and are then reverse-transcribed into genomic DNA by the associated intron-encoded protein. Target site recognition involves modifiable base-pairing interactions between the intron RNA and a >14-nucleotide region of the DNA target site, as well as fixed interactions between the protein and flanking regions. Here, we developed a highly efficient Escherichia coli genetic assay to determine detailed target site recognition rules for the Lactococcus lactis group II intron Ll.LtrB and to select introns that insert into desired target sites. Using human immunodeficiency virus-type 1 (HIV-1) proviral DNA and the human CCR5 gene as examples, we show that group II introns can be retargeted to insert efficiently into virtually any target DNA and that the retargeted introns retain activity in human cells. This work provides the practical basis for potential applications of targeted group II introns in genetic engineering, functional genomics, and gene therapy.     

Efficient trans-splicing of a yeast mitochondrial RNA group II intron implicates a strong 5' exon-intron interaction

The reaction mechanism for self-splicing introns requires the existence of a 5' exon binding site on the intron. Experimental evidence is now presented consistent with the existence of such a binding site by demonstrating efficient and accurate trans-self-splicing of a yeast mitochondrial group II intron. Partial and complete trans-splicing reactions take place in the absence of branch formation, part of the usual pathway of nuclear splicing and group II self-splicing. In addition to indicating the existence of a 5' exon binding site on the intron, the results have mechanistic implications for group II self-splicing and perhaps for nuclear splicing as well.     

Visualizing the higher order folding of a catalytic RNA molecule

The higher order folding process of the catalytic RNA derived from the self-splicing intron of Tetrahymena thermophila was monitored with the use of Fe(II)-EDTA-induced free radical chemistry. The overall tertiary structure of the RNA molecule forms cooperatively with the uptake of at least three magnesium ions. Local folding transitions display different metal ion dependencies, suggesting that the RNA tertiary structure assembles through a specific folding intermediate before the catalytic core is formed. Enzymatic activity, assayed with an RNA substrate that is complementary to the catalytic RNA active site, coincides with the cooperative structural transition. The higher order RNA foldings produced by Mg(II), Ca(II), and Sr(II) are similar; however, only the Mg(II)-stabilized RNA is catalytically active. Thus, these results directly demonstrate that divalent metal ions participate in general folding of the ribozyme tertiary structure, and further indicate a more specific involvement of Mg(II) in catalysis.     

草菇钙调磷酸酶催化亚基(Vv-CNA)的序列与表达

[目的]分析草菇钙调磷酸酶催化亚基(Vv-CNA)的序列与表达,为进一步探究草菇生长发育及子实体开伞分子调控机理提供参考.[方法]通过生物信息学分析确定Vv-CNA基因的组成及保守结构,将ORF Finder预测的草菇CNA氨基酸序列与从NCBI下载的其他真菌CNA氨基酸序列进行比对,并通过荧光定量PCR检测Vv-CNA基因在草菇不同生长时期的相对表达量.[结果]Vv-CNA基因编码区为2503 bp,包含10个内含子(其中I2、I8、I9和I10存在内含子保留现象),编码610个氨基酸,GenBank登录号KX463514.将Vv-CNA氨基酸序列与动物(人、小鼠)、植物(拟南芥、水稻)和真菌(曲霉、酵母等)的CNA氨基酸序列进行同源比对分析,结果发现Vv-CNA蛋白有3个最保守的结构域,与密褐褶菌(Gloeophyllum trabeum)的亲缘关系最近.Vv-CNA基因在草菇子实体的蛋形期和伸长期高表达,以蛋形期的相对表达量最高.[结论]Vv-CNA基因编码钙调磷酸酶催化亚基,其高表达有利于草菇菌柄的伸长.     

Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein

The removal of introns from eukaryotic messenger RNA precursors shares mechanistic characteristics with the self-splicing of certain introns, prompting speculation that the catalytic reactions of nuclear pre-messenger RNA splicing are fundamentally RNA-based. The participation of five small nuclear RNAs (snRNAs) in splicing is now well documented. Genetic analysis in yeast has revealed the requirement, in addition, for several dozen proteins. Some of these are tightly bound to snRNAs to form small nuclear ribonucleoproteins (snRNPs); such proteins may promote interactions between snRNAs or between an snRNA and the intron. Other, non-snRNP proteins appear to associate transiently with the spliceosome. Some of these factors, which include RNA-dependent adenosine triphosphatases, may promote the accurate recognition of introns.     

An ancient group I intron shared by eubacteria and chloroplasts

Introns have been found in the genomes of all major groups of organisms except eubacteria. The presence of introns in chloroplasts and mitochondria, both of which are of eubacterial origin, has been interpreted as evidence either for the recent acquisition of introns by organelles or for the loss of introns from their eubacterial progenitors. The gene for the leucine transfer RNA with a UAA anticodon [tRNALeu (UAA)] from five diverse cyanobacteria and several major groups of chloroplasts contains a single group I intron. The intron is conserved in secondary structure and primary sequence, and occupies the same position, within the UAA anticodon. The homology of the intron across chloroplasts and cyanobacteria implies that it was present in their common ancestor and that it has been maintained in their genomes for at least 1 billion years.     

Exon-intron organization in genes of earthworm and vertebrate globins

The structure of an invertebrate, intron-containing globin gene has been determined as part of a study of the evolution of hemoglobin. The gene encoding chain c of Lumbricus terrestris hemoglobin has the two-intron, three-exon structure characteristic of vertebrate globin genes, and the exact positions of the splice junctions are conserved. The two introns interrupting the coding sequence are longer than those of known hemoglobins but shorter than myoglobin introns. The gene encodes a secretory preglobin containing a 16-residue signal peptide, as expected for an extracellular hemoglobin. However, no intron separates the DNA encoding the signal sequence from that of the globin sequence. The 3' untranslated region of the Lumbricus gene is much longer than those of the genes for other hemoglobins and is similar to those found for myoglobins.     

扶桑绵粉蚧钙调蛋白基因的克隆与生物信息学分析

钙调蛋白(calmodulin,CaM)对生物体内多种Ca2+依赖的细胞功能和酶体系都有重要的调节作用。为研究扶桑绵粉蚧的信号转导受体蛋白,首次克隆了扶桑绵粉蚧钙调蛋白基因PsCaM的cDNA全长序列,其开放阅读框(ORF)包含447bp的片段,编码148个氨基酸。PsCaM基因由3个内含子和4个外显子组成。3个内含子的长度分别为73、81、72bp,分隔的4个外显子的长度分别为33、133、183、98bp。功能域分析结果显示:该蛋白具有2个EF-hand结构域,有13个Ca2+结合位点;该蛋白的理论等电点是6.21,属于稳定蛋白,且没有跨膜区域;通过同源建模获得了其蛋白的三维结构。多序列比较显示,PsCaM基因相对较保守。     

Structural evidence for a two-metal-ion mechanism of group I intron splicing

We report the 3.4 angstrom crystal structure of a catalytically active group I intron splicing intermediate containing the complete intron, both exons, the scissile phosphate, and all of the functional groups implicated in catalytic metal ion coordination, including the 2'-OH of the terminal guanosine. This structure suggests that, like protein phosphoryltransferases, an RNA phosphoryltransferase can use a two-metal-ion mechanism. Two Mg2+ ions are positioned 3.9 angstroms apart and are directly coordinated by all six of the biochemically predicted ligands. The evolutionary convergence of RNA and protein active sites on the same inorganic architecture highlights the intrinsic chemical capacity of the two-metal-ion catalytic mechanism for phosphoryl transfer.     

Control of gene expression by artificial introns in Saccharomyces cerevisiae

Artificial yeast introns that show cold-sensitive splicing have been constructed. These conditional introns can be inserted into a target gene as an "intron cassette" without disrupting the coding information, allowing expression of the gene to be cold sensitive. Insertion of these intron cassettes rendered the yeast URA3 gene cold sensitive in its expression. The advantage of this intron-mediated control system is that any gene can be converted to a controllable gene by simple insertion of an intron.     

Bacterial origin of a chloroplast intron: conserved self-splicing group I introns in cyanobacteria

A self-splicing group I intron has been found in the gene for a leucine transfer RNA in two species of Anabaena, a filamentous nitrogen-fixing cyanobacterium. The intron is similar to one that is found at the identical position in the same transfer RNA gene of chloroplasts of land plants. Because cyanobacteria were the progenitors of chloroplasts, it is likely that group I introns predated the endosymbiotic association of these eubacteria with eukaryotic cells.     

玫烟色棒束孢几丁质酶基因部分序列的克隆及分析

为了得到玫烟色棒束孢几丁质酶基因的保守区域,根据GenBank中几丁质酶基因的同源性序列设计简并引物,采用DNA和RNA分别作为模板扩增玫烟色棒束孢的几丁质酶保守区域。结果表明,DNA扩增的含有3个内含子,RNA扩增的不含内含子;所得到的氨基酸序列具有几丁质酶18族的典型特征,即1个酶作用活性位点和几丁质结合区。     

蓖麻WRKY14基因的鉴别与信息分析

基于已公布的蓖麻基因组序列,利用电子克隆技术纠正了拼接错误的WRKY基因序列,获得蓖麻WRKY14基因信息.结果表明:该基因含有3个内含子,编码318个氨基酸的多肽,理论相对分子质量为35.49×103,等电点为8.64,总平均亲水性为-0.803,不稳定系数为46.88,核定位,属于不稳定的亲水性核定位蛋白;该蛋白含有1个WRKY结构域,其基序为WRKYGQK_X13_C_X5_C_X23_H_X1_H,保守内含子位于199位的K和200位的V之间,可归为Ⅱ大类a亚类;RcWRKY14的启动子区含有多种激素和胁迫应答相关元件Rc WRKY14在叶片、花、胚乳和种子中都有表达,其中在花中的表达量最高,种子和叶片中次之,胚乳中最低.     

茶树PCP基因内含子的克隆与序列分析

利用GenBank中公布的茶树花粉壁蛋白基因（PCP）序列设计引物,采用PCR法扩增并测定茶树花粉壁蛋白基因内含子序列。在该基因中发现一个内含子,共207bp,A＋T碱基占67．63％,G＋C碱基占32．37％。经分析发现该内含子具有类似启动子的序列存在,可能对茶树花粉壁蛋白的特异表达有调控作用。     

Evolution of yeast noncoding RNAs reveals an alternative mechanism for widespread intron loss

The evolutionary forces responsible for intron loss are unresolved. Whereas research has focused on protein-coding genes, here we analyze noncoding small nucleolar RNA (snoRNA) genes in which introns, rather than exons, are typically the functional elements. Within the yeast lineage exemplified by the human pathogen Candida albicans, we find--through deep RNA sequencing and genome-wide annotation of splice junctions--extreme compaction and loss of associated exons, but retention of snoRNAs within introns. In the Saccharomyces yeast lineage, however, we find it is the introns that have been lost through widespread degeneration of splicing signals. This intron loss, perhaps facilitated by innovations in snoRNA processing, is distinct from that observed in protein-coding genes with respect to both mechanism and evolutionary timing.     

桑树WRKY转录因子的全基因组鉴定及生物信息学分析

[目的]明确桑树基因组中WRKY转录因子家族结构及其功能特征,为进一步揭示WRKY转录因子家族生物学功能提供科学依据.[方法]利用生物信息学方法对桑树WRKY转录因子的数目、类型、结构、系统进化关系、保守结构域和密码子使用偏性等进行全面分析.[结果]基于桑树全基因组蛋白数据库,共鉴定出55个桑树WRKY转录因子家族基因,占桑树基因总数(29261)的1.88%.桑树WRKY转录因子存在6种内含子数量类型及15种内含子相位类型,其中27个基因含有2个内含子,25个基因的相位类型为2-2型.保守结构域系统进化分析结果显示,桑树WRKY转录因子家族蛋白主要分为三大类(Ⅰ、Ⅱ和Ⅲ),Ⅰ类可分为ⅠN和ⅠC两个亚组,Ⅱ类根据聚类情况又可分为Ⅱa、Ⅱb、Ⅱc、Ⅱd和Ⅱe等5个亚组.桑树WRKY转录因子蛋白保守结构域分析发现有五类Motif的保守性较强,桑树WRKY转录因子蛋白中均包含C端Motif l,Ⅰ类蛋白同时含有N端Motif 3.桑树WRKY转录因子家族基因启动子区富含PBF(C2H2锌指因子)和AHL(拟南芥hook因子)元件.密码子使用偏性分析结果显示,桑树WRKY转录因子家族基因的有效密码子数(ENC)介于48.00~60.00,密码子第3位GC含量(GC3s)介于0.330~0.722,平均亲水性值(Gravy)均为负值;同义密码子相对使用度(RSCU)>1.000的密码子有29个,且以A(6个)或T(11个)结尾较G(4个)或C(8个)结尾的略多.[结论]桑树WRKY转录因子家族包含55个成员,内含子相位类型一致的同组成员可能来源于同一祖先基因,且与基因复制和基因组重排有关;蛋白序列高度保守,在植物抵御环境胁迫过程中发挥作用;基因密码子使用偏性较弱,主要受碱基突变选择压力影响.     

Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing

U6 is one of the five small nuclear RNA's (snRNA's) that are required for splicing of nuclear precursor messenger RNA (pre-mRNA). The size and sequence of U6 RNA are conserved among organisms as diverse as yeast and man, and so it has been proposed that U6 RNA functions as a catalytic element in splicing. A procedure for in vitro reconstitution of functional yeast U6 small nuclear ribonucleoproteins (snRNP's) with synthetic U6 RNA was applied in an attempt to elucidate the function of yeast U6 RNA. Two domains in U6 RNA were identified, each of which is required for in vitro splicing. Single nucleotide substitutions in these two domains block splicing either at the first or the second step. Invariably, U6 RNA mutants that block the first step of splicing do not enter the spliceosome. On the other hand, those that block the second step of splicing form a spliceosome but block cleavage at the 3' splice site of the intron. In both domains, the positions of base changes that block the second step of splicing correspond exactly to the site of insertion of pre-mRNA-type introns into the U6 gene of two yeast species, providing a possible explanation for the mechanism of how these introns originated and adding further evidence for the proposed catalytic role of U6 RNA.     

Ⅱ型内含子介导的基因打靶技术及应用前景

Ⅱ型内含子除具有核酶的特性之外,还可作为一种可移动的元件特异性地转移到基因组序列中,其转移过程需要内含子RNA和内含子编码蛋白质（IEP）来识别靶标位点,内含子已经作为一种新型的基因打靶工具应用于原核生物的基因插入失活和特定的基因敲除,并在真核生物基因组的功能基因组学和基因治疗中展现了广阔的应用前景。