Nucleotide sequence of KB cell 5S RNA

The nucleotide sequence of 5S RNA derived from KB carcinoma cell ribosomes has been determined. The molecule has a length of either 120 or 121 nucleotides with uridine at its 3'-terminus and guanylic acid at its 5'-terminus. If, in addition to Watson-Crick base-pairing, one accepts occasional base-pairing of guanylic acid to uridylic acid, long sequences of complementary nucleotides can be identified within the molecule. Two regions of the molecule contain sequences complementary to four or five bases in the pentanucleotide sequence guanylic acid, ribothymidylic acid, pseudouridylic acid, cytidylic acid, guanylic acid, which is common to most transfer RNA molecules. This is the first time the sequence of an animal-cell RNA has been determined.     

Heterogeneity of 5S RNA in fungal ribosomes

Neurospora crassa has at least seven types of 5S RNA genes (alpha, beta, gamma, epsilon, delta, zeta, and eta) with different coding regions. A high resolution gel electrophoresis system was developed to separate minor 5S RNA's from the major 5S RNA (alpha). A study of several Neurospora crassa strains, four other species in the genus Neurospora, members of two closely related genera, and three distantly related genera demonstrated that 5S RNA heterogeneity is common among fungi. In addition, different 5S RNA's are present in Neurospora ribosomes. The finding that fungal ribosomes are structurally heterogeneous suggests that ribosomes may be functionally heterogeneous as well.     

Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome

The splicing process, which removes intervening sequences from messenger RNA (mRNA) precursors is essential to gene expression in eukaryotic cells. This site-specific process requires precise sequence recognition at the boundaries of an intervening sequence, but the mechanism of this recognition is not understood. The splicing of mRNA precursors occurs in a multicomponent complex termed the spliceosome. Such an assembly of components is likely to play a key role in specifying those sequences to be spliced. In order to analyze spliceosome structure, a stringent approach was developed to obtain splicing complexes free of cellular contaminants. This approach is a form of affinity chromatography based on the high specificity of the biotin-streptavidin interaction. A minimum of three subunits: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles were identified in the 35S spliceosome structure, which also contains the bipartite RNA intermediate of splicing. A 25S presplicing complex contained only the U2 particle. The multiple subunit structure of the spliceosome has implications for the regulation of a splicing event and for its possible catalysis by ribozyme or ribozymes.     

U1-specific protein C needed for efficient complex formation of U1 snRNP with a 5' splice site

One of the functions of U1 small nuclear ribonucleoprotein (snRNP) in the splicing reaction of pre-mRNA molecules is the recognition of the 5' splice site. U1 snRNP proteins as well as base-pair interactions between U1 snRNA and the 5' splice site are important for the formation of the snRNP-pre-mRNA complex. To determine which proteins are needed for complex formation, the ability of U1 snRNPs gradually depleted of the U1-specific proteins C, A, and 70k to bind to an RNA molecule containing a 5' splice site sequence was studied in a nitrocellulose filter binding assay. The most significant effect was always observed when protein C was removed, either alone or together with other U1-specific proteins; the binding was reduced by 50 to 60%. Complementation of protein C-deficient U1 snRNPs with purified C protein restored their 5' splice site binding activity. These data suggest that protein C may potentiate the base-pair interaction between U1 RNA and the 5' splice site.     

Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome

Major structural changes occur in the spliceosome during its activation just before catalyzing the splicing of pre-messenger RNAs (pre-mRNAs). Whereas changes in small nuclear RNA (snRNA) conformation are well documented, little is known about remodeling of small nuclear ribonucleoprotein (snRNP) structures during spliceosome activation. Here, human 45S activated spliceosomes and a previously unknown 35S U5 snRNP were isolated by immunoaffinity selection and were characterized by mass spectrometry. Comparison of their protein components with those of other snRNP and spliceosomal complexes revealed a major change in protein composition during spliceosome activation. Our data also suggest that the U5 snRNP is dramatically remodeled at this stage, with the Prp19 complex and other factors tightly associating, possibly in exchange for other U5 proteins, and suggest that after catalysis the remodeled U5 is eventually released from the postsplicing complex as a 35S snRNP particle.     

U1 small nuclear RNA genes are subject to dosage compensation in mouse cells

Multiple copies of a gene that encodes human U1 small nuclear RNA were introduced into mouse C127 cells with bovine papilloma virus as the vector. For some recombinant constructions, the human U1 gene copies were maintained extrachromosomally on the viral episome in an unrearranged fashion. The relative abundance of human and mouse U1 small nuclear RNA varied from one cell line to another, but in some lines human U1 RNA accounted for as much as one-third of the total U1. Regardless of the level of human U1 expression, the total amount of U1 RNA (both mouse and human) in each cell line was nearly the same relative to endogenous mouse 5S or U2 RNA. This result was obtained whether measurements were made of total cellular U1 or of only the U1 in small nuclear ribonucleoprotein particles that could be precipitated with antibody directed against the Sm antigen. The data suggest that the multigene families encoding mammalian U1 RNA are subject to some form of dosage compensation.     

Localization of 5S RNA genes on Drosophila chromosomes by RNA-DNA hybridization

[(3)H]RNA with a high specific activity was prepared from larvae of Drosophila melanogaster grown 4 days in contact with [(3)H]uridine. Purified tritiated 5S RNA was annealed to the DNA of polytene chromosomes, which had been denatured in formamide. The 5S RNA genes are placed within the region 56E-F of the right arm of chromosome 2. This localization was determined from autoradiographs, where the radioactivity from hybrids of [(3)H]RNA and DNA was confined to the 56E-F segment.     

Synthesis of 5S and 4S RNA in metaphase-arrested HeLa cells

The continued synthesis of both 5S and 4S RNA in metaphase-arrested HeLa cells is demonstrated; 5S RNA is apparently synthesized at approximately 74 percent of the interphase rate, while 4S RNA is synthesized at approximately one-third the rate. The ratio of uridine incorporation to RNA methylation is used to correct for the alteration in the specific activity of the pyrimidine pool during metaphase arrest.     

NMR detection of structures in the HIV-1 5'-leader RNA that regulate genome packaging

The 5'-leader of the HIV-1 genome regulates multiple functions during viral replication via mechanisms that have yet to be established. We developed a nuclear magnetic resonance approach that enabled direct detection of structural elements within the intact leader (712-nucleotide dimer) that are critical for genome packaging. Residues spanning the gag start codon (AUG) form a hairpin in the monomeric leader and base pair with residues of the unique-5' region (U5) in the dimer. U5:AUG formation promotes dimerization by displacing and exposing a dimer-promoting hairpin and enhances binding by the nucleocapsid (NC) protein, which is the cognate domain of the viral Gag polyprotein that directs packaging. Our findings support a packaging mechanism in which translation, dimerization, NC binding, and packaging are regulated by a common RNA structural switch.     

Crystal structure of the ribosome at 5.5 A resolution

We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution. All of the 16S, 23S, and 5S ribosomal RNA (rRNA) chains, the A-, P-, and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 angstrom movements associated with tRNA translocation with intersubunit movement.     

S5-95U可编程控制器在热媒炉控制系统中的应用

介绍了东黄输油管道热煤加热炉中应用西门子Ｓ５－９５Ｕ自动控制系统情况。利用Ｓ５－９５Ｕ可编顺控制器开发的９５Ｕ炉控系统及炉控人机操作界面，优化了控制结构，使加热系统安全运行，收到了良好效果。     

Reversal of creatine kinase translational repression by 3' untranslated sequences

A subline of U937 cells (U937D) was obtained in which creatine kinase B (CK-B) messenger RNA was present and bound to ribosomes, but CK activity was undetectable. Transformation of U937D cells with retrovirus vectors that contain the 3' untranslated region (3' UTR) of CK-B messenger RNA exhibited CK activity with no change in abundance of CK-B mRNA. The 3' UTR formed a complex in vitro with a component of S100 extracts from wild-type cells. This binding activity was not detectable in S100 extracts from cells that expressed CK activity after transformation with the 3' UTR-containing vector. These results suggest that translation of CK-B is repressed by binding of a soluble factor or factors to the 3' UTR.     

山羊卵巢富集chi-miR-100f-5p的鉴定与表达分析

【目的】探讨山羊卵巢中富集的miR-100及预测的靶基因与山羊卵巢功能及卵泡发育的潜在关系,为山羊繁殖机理研究提供数据。【方法】 以山羊卵巢组织为材料,通过miRNA的分离、克隆及测序得到山羊卵巢中表达的miR-100,采用RT-qPCR进行表达检测,用生物信息学方法预测miR-100的靶基因及其功能。【结果】① 山羊卵巢的测序结果中,一段成熟体为21nt 的序列与果蝇dan-miR-100高度同源,将其命名为chi-miR-100f-5p,其前体位于基因组负（-）链上,长度为59 bp,定位于山羊CM000899.1:33 997 894—33 997 914序列;② chi-miR-100f-5p 在山羊卵巢、垂体及心脏等组织中均有表达,说明此miRNA不是卵巢组织所特有;心脏和垂体组织中均有较高表达,其次是卵巢,而在肝脏、脾脏、肺及肾脏中表达显著低于卵巢（P ＜0.01）;在单产羔内蒙古绒山羊卵巢中chi-miR-100f-5p的表达极显著高于高繁殖力大足黑山羊卵巢（P＜0.01）;③ chi-miR-100f-5p主要靶作用于整合蛋白β1亚基基因及垂体特异性转录因子1基因,可能参与整合蛋白信号通路,促进垂体内分泌细胞的生长与分化,调节下丘脑-垂体-性腺轴激素分泌。【结论】山羊卵巢中新发现的chi-miR-100f-5p 可能是与繁殖调控有关的重要候选miRNAs 之一。     

Small nuclear RNA U2 is base-paired to heterogeneous nuclear RNA

Eukaryotic cells contain a set of low molecular weight nuclear RNA's. One of the more abundant of these is termed U2 RNA. The possibility that U2 RNA is hydrogen-bonded to complementary sequences in other nuclear RNA's was investigated. Cultured human (HeLa) cells were treated with a psoralen derivative that cross-links RNA chains that are base-paired with one another. High molecular weight heterogeneous nuclear RNA was isolated under denaturing conditions, and the psoralen cross-links were reversed. Electrophoresis of the released RNA and hybridization with a human cloned U2 DNA probe revealed that U2 is hydrogen-bonded to complementary sequences in heterogeneous nuclear RNA in vivo. In contrast, U2 RNA is not base-paired with nucleolar RNA, which contains the precursors of ribosomal RNA. The results suggest that U2 RNA participates in messenger RNA processing in the nucleus.     

An essential signaling role for the m3G cap in the transport of U1 snRNP to the nucleus

The major small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4 + U6, and U5 have to be transported from the cytoplasm, where they are synthesized, to the nucleus, where they splice pre-messenger RNAs. Since the free core snRNP proteins in the cytoplasm do not enter the nucleus on their own, the nuclear location signal must either reside on the snRNA or be created as a result of snRNA-protein interaction. Here the involvement by the 5'-terminal cap of snRNA molecules in the nucleo-cytoplasmic transport of UsnRNPs has been studied by microinjection of synthetic U1 RNA molecules into frog oocytes; the U1 RNA bore either the normal cap (m3G) or a chemical derivative. Antibodies in the cytoplasm against the m3G cap inhibited the nuclear uptake of U1 snRNP. U1 RNA that was uncapped or contained an unnatural ApppG cap did not enter the nucleus, even though it carried a normal complement of protein molecules. When the ribose ring of the m3G cap was oxidized with periodate, nuclear transport of U1 snRNPs was severely inhibited. Finally, microinjection of m3G cap alone (but not m7G cap) into oocytes severely inhibited the transport of U1 snRNPs to the nucleus. These data suggest that one step in the nuclear uptake of U1 snRNPs involves the m3G cap structure.     

三-(五甲基环戊二烯基)稀土有机配合物中的体积诱导效应

讨论了三-(五甲基环戊二烯基)稀土有机配合物的反应性质,探讨了它们的反应性能与体积效应的关系,发现随着体积效应的增大,三-(五甲基环戊二烯基)稀土有机配合物能发生体积诱导还原反应。     

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.     

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.