The design and catalytic properties of a simplified ribonuclease P RNA

Ribonuclease P (RNase P) RNA is the catalytic moiety of the ribonucleoprotein enzyme that removes precursor sequences from the 5' ends of pre-transfer RNAs in eubacteria. Phylogenetic variation according to recently proposed secondary structure models was used to identify structural elements of the RNase P RNA that are dispensable for catalysis. A simplified RNase P RNA that consists only of evolutionarily conserved features was designed, synthesized, and characterized. Although the simplified RNA (Min 1 RNA) is only 263 nucleotides in length, in contrast to the 354 to 417 nucleotides of naturally occurring RNase P RNAs, its specificity of pre-tRNA cleavage is identical to that of the native enzymes. Moreover, the catalytic efficiencies of the Min 1 RNA and the native RNA enzymes are similar. These results focus the search for the catalytic elements of RNase P RNAs to their conserved structure.     

Structural basis for double-stranded RNA processing by Dicer

The specialized ribonuclease Dicer initiates RNA interference by cleaving double-stranded RNA (dsRNA) substrates into small fragments about 25 nucleotides in length. In the crystal structure of an intact Dicer enzyme, the PAZ domain, a module that binds the end of dsRNA, is separated from the two catalytic ribonuclease III (RNase III) domains by a flat, positively charged surface. The 65 angstrom distance between the PAZ and RNase III domains matches the length spanned by 25 base pairs of RNA. Thus, Dicer itself is a molecular ruler that recognizes dsRNA and cleaves a specified distance from the helical end.     

Autoreactive epitope defined as the anticodon region of alanine transfer RNA

Autoantibodies to aminoacyl-transfer RNA (tRNA) synthetases are common in the human autoimmune diseases polymyositis and dermatomyositis. Sera of the PL-12 specificity contain separate antibodies reacting with alanyl-tRNA synthetase and alanine tRNA (tRNAAla). The antibodies to tRNA recognize at least six distinguishable human tRNAAla species grouped into two sequence families. The antibody-reactive determinants on the tRNA were identified through ribonuclease protection and oligonucleotide binding experiments. The antibody binding site is a seven- to nine-nucleotide sequence containing the anticodon loop and requires an intact anticodon. No requirement for anticodon stem structure or sequence is observed, although the 5' portion of the stem is protected from nuclease attack. Antibodies from several patients appear to share the same specificitym, indicating that the antibodies are induced by a unique sequence feature in the immunogen.     

Binding of the human Prp31 Nop domain to a composite RNA-protein platform in U4 snRNP

Although highly homologous, the spliceosomal hPrp31 and the nucleolar Nop56 and Nop58 (Nop56/58) proteins recognize different ribonucleoprotein (RNP) particles. hPrp31 interacts with complexes containing the 15.5K protein and U4 or U4atac small nuclear RNA (snRNA), whereas Nop56/58 associate with 15.5K-box C/D small nucleolar RNA complexes. We present structural and biochemical analyses of hPrp31-15.5K-U4 snRNA complexes that show how the conserved Nop domain in hPrp31 maintains high RNP binding selectivity despite relaxed RNA sequence requirements. The Nop domain is a genuine RNP binding module, exhibiting RNA and protein binding surfaces. Yeast two-hybrid analyses suggest a link between retinitis pigmentosa and an aberrant hPrp31-hPrp6 interaction that blocks U4/U6-U5 tri-snRNP formation.     

甜菜杂种优势与核糖核酸酶同工酶的关系

通过甜菜杂交组合的核糖核酸酶（ＲＮａｓｅ）同工酶与杂种优势关系的研究表明，一对真叶期和六叶期ＲＮａｓｅ同工酶的酶蛋白含量，在其组合内均以杂种１代为最低，呈负优势，与块根含糟率达极显著负相关，是选育丰产高糖型优势杂种的重要生理指标。     

Mutagenic processing of ribonucleotides in DNA by yeast topoisomerase I

The ribonuclease (RNase) H class of enzymes degrades the RNA component of RNA:DNA hybrids and is important in nucleic acid metabolism. RNase H2 is specialized to remove single ribonucleotides [ribonucleoside monophosphates (rNMPs)] from duplex DNA, and its absence in budding yeast has been associated with the accumulation of deletions within short tandem repeats. Here, we demonstrate that rNMP-associated deletion formation requires the activity of Top1, a topoisomerase that relaxes supercoils by reversibly nicking duplex DNA. The reported studies extend the role of Top1 to include the processing of rNMPs in genomic DNA into irreversible single-strand breaks, an activity that can have distinct mutagenic consequences and may be relevant to human disease.     

小麦条锈病罹病叶片核酸水解酶活力的变化

小麦初生叶接种条锈菌后，亲和反应寄主叶片ＲＮａｓｅ活性，分别在潜育期和产孢期呈双峰型增长；不亲和反应叶片ＲＮａｓｅ活性在侵染初期高于健康对照，但低于亲和性反应，其后随着病程发展，近免疫反应叶片ＲＮａｓｅ活性与对照相同，而中度抗病反应叶片也呈双峰增长，峰高及峰延续时间不同于亲和性反应。条锈菌侵染对ＤＮａｓｅ活性影响较小，亲和反应叶片仅在产孢阶段ＤＮａｓｅ活性有所增强，中度抗病反应叶片在显症时活性增强。     

Expression and characterization of the trans-activator of HTLV-III/LAV virus

The human T-lymphotropic retrovirus HTLV-III/LAV encodes a trans-activator that increases viral gene expression. We expressed this trans-activator in animal cells and studied its structural and functional characteristics. The putative trans-activator protein was immunoprecipitated from overproducing stable cell lines and shown to migrate as a 14-kilodalton polypeptide on sodium dodecyl sulfate-polyacrylamide gels. S1 nuclease mapping experiments showed that the trans-activator increases the levels of steady-state messenger RNA transcribed from the viral long terminal repeat promoter. Sequences within the R region of the HTLV-III/LAV long terminal repeat are essential for trans-activation. Quantitations of messenger RNA and protein showed that the protein increase was greater than the messenger RNA increase in CV1 and HeLa cells, indicating that more than one mechanism was responsible for the trans-activation and that cell type-specific factors may determine the final level of trans-activation.     

Antibodies to human c-myc oncogene product: evidence of an evolutionarily conserved protein induced during cell proliferation

Antisera to a synthetic c-myc peptide and to c-myc antigens synthesized from various portions of the human gene expressed in Escherichia coli were used in order to characterize the protein product of the human c-myc oncogene. Although the deduced molecular weight of the human c-myc protein is 49,000, these antisera precipitate a protein from human cells that migrates in sodium dodecyl sulfate-polyacrylamide gel as if its molecular weight were 65,000. In addition, the mouse c-myc protein, whether synthesized in cells or in a cell-free system directed by pure, synthetic messenger RNA, has analogous properties and is immunoprecipitated by the antiserum to the human c-myc protein. Similar proteins are immunoprecipitated from monkey, rat, hamster, and frog cells, suggesting evolutionary conservation of antigenic structure of the c-myc protein among vertebrates. In addition, and in a manner consistent with the behavior of its messenger RNA, the immunoprecipitable c-myc protein is sharply induced by the action of mitogens on resting human T cells.     

Two novel classes of small ribonucleoproteins detected by antibodies associated with lupus erythematosus

The RNP and Sm antigens recognized by lupus erythematosus antibodies are located on discrete particles containing single small nuclear RNA's complexed with proteins. The antigens Ro and La are also on ribonucleoproteins. The small RNA's in ribonucleoproteins with Ro are discrete, like those associated with RNP and Sm; in contrast, ribonucleoproteins with La contain a striking highly banded spectrum of small RNA's from uninfected cells as well as virus-associated RNA from adenovirus-infected cells.     

Autolytic processing of dimeric plant virus satellite RNA

Associated with some plant viruses are small satellite RNA's that depend on the plant virus to provide protective coat protein and presumably at least some of the proteins necessary for satellite RNA replication. Multimeric forms of the satellite RNA of tobacco ringspot virus are probable in vivo precursors of the monomeric satellite RNA. Evidence is presented for the in vitro autolytic processing of dimeric and trimeric forms of this satellite RNA. The reaction generates biologically active monomeric satellite RNA, apparently is reversible to form dimeric RNA from monomeric RNA, and does not require an enzyme for its catalysis.     

Specific interactions in RNA enzyme-substrate complexes

Analysis of crosslinked complexes of M1 RNA, the catalytic RNA subunit of ribonuclease P from Escherichia coli, and transfer RNA precursor substrates has led to the identification of regions in the enzyme and in the substrate that are in close physical proximity to each other. The nucleotide in M1 RNA, residue C92, which participates in a crosslink with the substrate was deleted and the resulting mutant M1 RNA was shown to cleave substrates lacking the 3' terminal CCAUCA sequence at sites several nucleotides away from the normal site of cleavage. The presence or absence of the 3' terminal CCAUCA sequence in transfer RNA precursor substrates markedly affects the way in which these substrates interact with the catalytic RNA in the enzyme-substrate complex. The contacts between wild-type M1 RNA and its substrate are in a region that resembles part of the transfer RNA "E" (exit) site in 23S ribosomal RNA. These data demonstrate that in RNA's with very different cellular functions, there are domains with similar structural and functional properties and that there is a nucleotide in M1 RNA that affects the site of cleavage by the enzyme.     

拟南芥DCL1启动子的分离及其表达特性分析

Dicer酶是类似于RNase Ⅲ的核酸内切酶,能够特异性地将双链RNA剪切成约20 nt的小RNA,在基因沉默途径中起到非常重要的作用。从拟南芥中分离出DCL1基因上游启动子序列2 kb及DCL1基因的5′端1 kb序列,构建了含有该启动子和GUS报告基因的植物表达载体,通过农杆菌介导法转化拟南芥,并对转基因植株进行GUS 组织化学染色及荧光定量分析,结果表明,在DCL1基因启动子的驱动下,报告基因GUS主要在拟南芥的叶片中表达,在幼嫩的叶片及茎尖中表达量也比较高,茎中的表达量较低,在根中只有微量的表达。     

R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway

The RNA interference (RNAi) pathway is initiated by processing long double-stranded RNA into small interfering RNA (siRNA). The siRNA-generating enzyme was purified from Drosophila S2cells and consists of two stoichiometric subunits: Dicer-2(DCR-2) and a previously unknown protein that we named R2D2. R2D2 is homologous to the Caenorhabditis elegans RNAi protein RDE-4. Association with R2D2 does not affect the enzymatic activity of DCR-2. Rather, the DCR-2/R2D2 complex, but not DCR-2 alone, binds to siRNA and enhances sequence-specific messenger RNA degradation mediated by the RNA-initiated silencing complex (RISC). These results indicate that R2D2 bridges the initiation and effector steps of the Drosophila RNAi pathway by facilitating siRNA passage from Dicer to RISC.     

A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans

An early event in RNA interference (RNAi) is the cleavage of the initiating double-stranded RNA (dsRNA) to short pieces, 21 to 23 nucleotides in length. Here we describe a null mutation in dicer-1 (dcr-1), a gene proposed to encode the enzyme that generates these short RNAs. We find that dcr-1(-/-) animals have defects in RNAi under some, but not all, conditions. Mutant animals have germ line defects that lead to sterility, suggesting that cleavage of dsRNA to short pieces is a requisite event in normal development.     

猪链球菌2型溶菌酶释放蛋白诱导血管内皮细胞融合

 猪链球菌2型（SS2）主要引起人和猪的脑膜炎，但其毒力因子溶菌酶释放蛋白(MRP)对构成血脑屏障的微血管内皮细胞有何致病作用迄今不明。为此分离仔兔脾微血管内皮细胞（SMEC），纯化后用SV40-T抗原转化后用作试验的细胞模型。将单层SMEC和电泳纯MRP溶液共孵育，染色后，光镜观察，发现MRP可诱导SMEC发生两种典型形态学变化：致密细胞单层中出现巨大空洞而呈网状；空洞内有细胞融合，形成多核巨细胞，随后巨细胞核极度浓缩释出，巨细胞消失。研究结果表明， MRP单独足以破坏大脑的血管内皮细胞屏障。     

Quantitation of RNA tumor viruses and viruslike particles in human milk by hybridization to polyadenylic acid sequences

RNA tumor viruses and viruslike particles from human milk are quantitated by hybridization of the polyadenylic acid regions in their 60S to 70S RNA to radioactive polyribouridylic acid of known specific activity. The length of the polyadenylic acid region in the 60S to 70S RNA of the human milk particle is identical to that of the known oncogenic RNA viruses.     

Human insulin-degrading enzyme shares structural and functional homologies with E. coli protease III

A proteinase with high affinity for insulin has been proposed to play a role in the cellular processing of this hormone. A complementary DNA (cDNA) coding for this enzyme has been isolated and sequenced. The deduced amino acid sequence of the enzyme contained the sequences of 13 peptides derived from the isolated protein. The cDNA could be transcribed in vitro to yield a synthetic RNA that in cell-free translations produced a protein that coelectrophoresed with the native proteinase and could be immunoprecipitated with monoclonal antibodies to this enzyme. The deduced sequence of this proteinase did not contain the consensus sequences for any of the known classes of proteinases (that is, metallo, cysteine, aspartic, or serine), but it did show homology to an Escherichia coli proteinase (called protease III), which also cleaves insulin and is present in the periplasmic space. Thus, these two proteins may be members of a family of proteases that are involved in intercellular peptide signaling.