Protein displacement by DExH/D "RNA helicases" without duplex unwinding

Members of the DExH/D superfamily of nucleic acid-activated nucleotide triphosphatases are essential for virtually all aspects of RNA metabolism, including pre-messenger RNA splicing, RNA interference, translation, and nucleocytoplasmic trafficking. Physiological substrates for these enzymes are thought to be regions of double-stranded RNA, because several DExH/D proteins catalyze strand separation in vitro. These "RNA helicases" can also disrupt RNA-protein interactions, but it is unclear whether this activity is coupled to duplex unwinding. Here we demonstrate that two unrelated DExH/D proteins catalyze protein displacement independently of duplex unwinding. Therefore, the essential functions of DExH/D proteins are not confined to RNA duplexes but can be exerted on a wide range of ribonucleoprotein substrates.     

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.     

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.     

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.     

基于4D label-free技术的水稻成熟种子蛋白质组学研究

【目的】从蛋白水平揭示水稻种子成熟的分子基础，探究调控水稻种子成熟的关键蛋白和代谢通路。【方法】选用授粉后30 d的成熟水稻种子，利用4D label-free定量蛋白质组学进行质谱鉴定，通过生物信息学技术分析蛋白的亚细胞定位、结构域、GO注释和KEGG通路注释。【结果】总共鉴定了3 484个种子成熟期的蛋白，相对分子质量大多在10 000～100 000之间，主要分布于细胞质、细胞核、叶绿体、线粒体和质膜上；结构域主要涉及蛋白质翻译的RNA识别基序和蛋白磷酸化修饰的蛋白激酶结构域；GO分析表明，成熟种子的蛋白主要参与了细胞过程和代谢过程，主要涉及催化活性和结合等功能，大多分布在细胞、细胞组分、细胞器和细胞膜等部位；KEGG分析发现，蛋白主要富集在核糖体、内质网中的蛋白质加工、氧化磷酸化和糖酵解等途径，推测蛋白质的翻译、加工和能量代谢是水稻种子成熟期的主要分子事件；进一步鉴定了脱落酸(Abscisic acid,ABA)信号和吲哚乙酸(Indoleacetic acid,IAA)代谢的相关蛋白，同时也发现了NAC(NAM、ATAF1/2和CUC2)家族的转录因子。【结论】贮藏物质的积累...     

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.     

Template activity of RNA from antibody-producing tissues

An RNA fraction, which represents a small percentage of cellular RNA and which has the characteristics of nuclear messenger RNA, has been isolated from the spleen and lymph nodes of immunized rats by successive phenol extractions of these tissues at increasing temperatures. This fraction increased the amount of protein synthesized in a cell-free extract of Escherichia coli as much as 35 times and directed the synthesis of proteins different from those of E. coli.     

Correlated measurements of DNA, RNA, and protein in individual cells by flow cytometry

A cytochemical method was developed to differentially stain cellular DNA, RNA, and proteins with fluorochromes Hoechst 33342, pyronin Y, and fluorescein isothiocyanate, respectively. The fluorescence intensities, reflecting the DNA, RNA, and protein content of individual cells, were measured in a flow cytometer after sequential excitation by three lasers tuned to different excitation wavelengths. The method offers rapid analysis of changes in the cellular content of RNA and protein as well as in the RNA-protein, RNA-DNA, and protein-DNA ratios in relation to cell cycle position for large cell populations. An analysis of cycling cell populations (exponentially growing CHO cultures) and noncycling CHO cells arrested in the G1 phase by growth in isoleucine-free medium demonstrated the potential of the technique.     

RNA mimics of green fluorescent protein

Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various applications, but a comparable approach for fluorescently tagging RNA is lacking. Here, we describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNA-fluorophore complexes create a palette that spans the visible spectrum. An RNA-fluorophore complex, termed Spinach, resembles enhanced GFP and emits a green fluorescence comparable in brightness with fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach for genetic encoding of fluorescent RNAs.     

Acylation of proteins with myristic acid occurs cotranslationally

Several proteins of viral and cellular origin are acylated with myristic acid early during their biogenesis. To investigate the possibility that myristylation occurred cotranslationally, the BC3H1 muscle cell line, which contains a broad array of myristylated proteins, was pulse-labeled with [3H]myristic acid. Nascent polypeptide chains covalently associated with transfer RNA were isolated subsequently by ion-exchange chromatography. [3H]Myristate was attached to nascent chains through an amide linkage and was identified by thin-layer chromatography after its release from nascent chains by acid methanolysis. Inhibition of cellular protein synthesis with puromycin resulted in cessation of [3H]myristate-labeling of nascent chains, in agreement with the dependence of this modification on protein synthesis in vivo. These data represent a direct demonstration that myristylation of proteins is a cotranslational modification.     

Assembly of functional U1 and U2 human-amphibian hybrid snRNPs in Xenopus laevis oocytes

Oligonucleotides complementary to regions of U1 and U2 small nuclear RNAs (snRNAs), when injected into Xenopus laevis oocytes, rapidly induced the specific degradation of U1 and U2 snRNAs, respectively, and then themselves were degraded. After such treatment, splicing of simian virus 40 (SV40) late pre-mRNA transcribed from microinjected viral DNA was blocked in oocytes. If before introduction of SV40 DNA into oocytes HeLa cell U1 or U2 snRNAs were injected and allowed to assemble into small nuclear ribonucleoprotein particle (snRNP)-like complexes, SV40 late RNA was as efficiently spliced as in oocytes that did not receive U1 or U2 oligonucleotides. This demonstrates that oocytes can form fully functional hybrid U1 and U2 snRNPs consisting of human snRNA and amphibian proteins.     

Cytokine-induced expression of HIV-1 in a chronically infected promonocyte cell line

A model system for cytokine-induced up-regulation of human immunodeficiency virus type 1 (HIV-1) expression in chronically infected promonocyte clones was established. The parent promonocyte cell line U937 was chronically infected with HIV-1 and from this line a clone, U1, was derived. U1 showed minimal constitutive expression of HIV-1, but virus expression was markedly up-regulated by a phytohemagglutinin-induced supernatant containing multiple cytokines and by recombinant granulocyte/macrophage colony-stimulating factor alone. Recombinant interleukin-1 (IL-1), IL-2, interferon-gamma, and tumor necrosis factor-alpha did not up-regulate virus expression. Concomitant with the cytokine-induced up-regulation of HIV-1, expression of membrane-bound IL-1 beta was selectively induced in U1 in the absence of induction of other surface membrane proteins. This cytokine up-regulation of IL-1 beta was not seen in the uninfected parent U937 cell line. These studies have implications for the understanding of the mechanism of progression from a latent or low-level HIV-1 infection to a productive infection with resulting immunosuppression. In addition, this model can be used to delineate the potential mechanisms whereby HIV-1 infection regulates cellular gene expression.     

稻曲病菌侵染水稻颖花的酵母双杂交cDNA文库构建与应用

【目的】近年来稻曲病日益严重,但目前对稻曲病菌(Ustilaginoidea virens)与水稻相互作用机制仍不清楚。论文旨在利用水稻感病颖花材料构建酵母双杂交文库并筛选稻曲病菌效应因子互作蛋白,为解析稻曲病菌侵染水稻机制提供理论基础。【方法】以感病水稻Chuannong H2S为试验材料,在水稻破口前5—7 d左右,从水稻穗中上部接种PSB摇培7 d的稻曲病菌荧光标记菌株P4,接种13 d后取颖花进行激光共聚焦观察,收集感病颖花。提取感病颖花总RNA,使用含有Oligo(dT)的接头引物反转录cDNA第一链,并PCR扩增ds cDNA,通过琼脂糖凝胶电泳切胶回收ds cDNA片段,与载体p GADT7-Rec进行同源重组,转化酵母菌株Y187构建酵母双杂交cDNA文库。然后用稻曲病菌效应因子Uv_1261基因构建诱饵载体,将诱饵载体转化入Y2HGold酵母菌株,通过酵母双杂交自激活验证后以该诱饵载体筛选酵母双杂交文库,最终在SD/-Ade/-His/-Leu/-Trp/X-α-Gal平板上筛选出生长状况良好的蓝色单菌落,经测序得到候选互作蛋白的序列,通过NCBI在线网站进行Blast分析和Uniprot在线网站进行gene ontology(GO)注释。【结果】经检测,文库滴度为5.7×108 cfu/mL,平均插入片段大小为750 bp,表明稻曲病菌侵染水稻颖花cDNA文库质量较高。用Uv_1261构建诱饵载体,转化Y2HGold后,转化菌可在SD/-Trp、SD/-Trp/X-α-Gal平板上生长,不能在SD/-Trp/X-α-Gal/Ab A平板上生长,表明Uv_1261无自激活现象。以该诱饵载体筛选文库,对筛选到的候选互作蛋白进行测序验证并Blast分析获得了56个来自稻曲病菌或水稻的候选互作蛋白,其中有28个候选互作蛋白来自稻曲病菌,有16个来自水稻,以及12个未知蛋白。经GO注释显示,来自稻曲病菌中的候选互作蛋白参与了17个生物过程,包括翻译、代谢过程、氧化还原及胞内氨基酸合成等;分子功能包括金属离子结合活性、水解酶活性及ATP结合活性等;细胞组分包括核糖体、细胞质及线粒体等。来自水稻中的候选互作蛋白参与了11个生物过程,包括蛋白质去磷酸化、糖代谢及翻译等;分子功能包括金属离子结合活性、核苷酸结合活性及转移酶活性等;细胞组分包括核糖体、膜及细胞核等。【结论】该cDNA文库质量较好,能成功地用于稻曲病菌效应蛋白的互作蛋白筛选,可为研究稻曲病菌与水稻相互作用的分子机制提供重要资源。     

Saccharomyces cerevisiae has a U1-like small nuclear RNA with unexpected properties

Previous experiments indicated that only a small subset of the approximately equal to 24 small nuclear RNAs (snRNAs) in Saccharomyces cerevisiae have binding sites for the Sm antigen, a hallmark of metazoan small nuclear ribonucleoproteins (snRNPs) involved in pre-messenger RNA splicing. Antibodies from human serum to Sm proteins were used to show that four snRNAs (snR7, snR14, snR19, and snR20) can be immunoprecipitated from yeast extracts. Three of these four, snR7, snR14, and snR20, have been shown to be analogs of mammalian U5, U4, and U2, respectively. Several regions of significant homology to U1 (164 nucleotides) have now been found in cloned and sequenced snR19 (568 nucleotides). These include ten out of ten matches to the 5' end of U1, the site known to interact with the 5' splice site of mammalian introns. Surprisingly, the precise conservation of this sequence precludes perfect complementarity between snR19 and the invariant yeast 5' junction (GTATGT), which differs from the mammalian consensus at the fourth position (GTPuAGT).     

Structural biology. The ribosome is a ribozyme

Ribosomes, the cellular factories that manufacture proteins, contain both RNA and protein, but exactly how all of the different ribosomal components contribute to protein synthesis is still not clear. Now, as Thomas Cech explains in his Perspective, atomic resolution of the structure of the large ribosomal subunit reveals that, as predicted by those convinced of a prebiotic RNA world, RNA is the catalytic component with proteins being the structural units that support and stabilize it (Ban et al., Nissen et al., Muth et al.).     

Reprogramming cellular behavior with RNA controllers responsive to endogenous proteins

Synthetic genetic devices that interface with native cellular pathways can be used to change natural networks to implement new forms of control and behavior. The engineering of gene networks has been limited by an inability to interface with native components. We describe a class of RNA control devices that overcome these limitations by coupling increased abundance of particular proteins to targeted gene expression events through the regulation of alternative RNA splicing. We engineered RNA devices that detect signaling through the nuclear factor κB and Wnt signaling pathways in human cells and rewire these pathways to produce new behaviors, thereby linking disease markers to noninvasive sensing and reprogrammed cellular fates. Our work provides a genetic platform that can build programmable sensing-actuation devices enabling autonomous control over cellular behavior.     

A mammalian H+ channel generated through alternative splicing of the NADPH oxidase homolog NOH-1

Voltage-gated proton (H+) channels are found in many human and animal tissues and play an important role in cellular defense against acidic stress. However, a molecular identification of these unique ion conductances has so far not been achieved. A 191-amino acid protein is described that, upon heterologous expression, has properties indistinguishable from those of native H+ channels. This protein is generated through alternative splicing of messenger RNA derived from the gene NOH-1 (NADPH oxidase homolog 1, where NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate).     

转化生长因子α对人肠上皮细胞增殖、细胞总RNA和总蛋白质含量的影响

主要探讨转化生长因子-α(Transforming Growth Factorα,TGF-α)对人结肠细胞生长、增殖以及细胞总RNA和总蛋白质含量的影响。以人结肠腺癌细胞(Adenocarcinoma,LoVo)为试验模型,分别添加重组人TGF-α0.1、1(接近人乳TGF-α含量最低值)、10(接近人乳TGF-α含量最高值)[1]和100ng·mL-1后,观察细胞增殖能力、细胞总RNA和总蛋白质含量变化。添加TGF-α24h后,细胞较对照有明显的增殖现象,且增值率随细胞浓度增加而呈上升趋势,并在10ng·mL-1时达到最大(P<0.01)。细胞总RNA含量随TGF-α浓度增加而上升为控制组的1.67倍(P<0.05)至4.20倍(P<0.01)。而100ng·mL-1组细胞总蛋白质含量明显高于控制组和其他各剂量组。为对照组的1.28倍(P<0.05)。TGF-α能够促进人肠上皮细胞细胞增殖且具有剂量依赖性,TGF-α同样能够促进人结肠上皮细胞RNA含量的增加和蛋白质的合成。