Stable RNA hairpins in 88 coding regions of human mRNA

RNA hairpins containing UNCG, GNRA, CUUG (N = A, U, C or G, R = G or A) loops are unusually thermodynamic stable and conserved structures. The structural features of these hairpin loops are very special, and they play very important roles in vivo. They are prevalent in rRNA, catalytic RNA and non-coding mRNA. However, the 5' C(UUCG)G 3' hairpin is not found in the folding structure of 88 human mRNA coding regions. It is also different from rRNA in that there is no preference for certain sequences among tetraloops in these 88 mRNA folding structures.

Statistical correlation between protein secondary structure and messenger RNA stem-loop structure

Anew integrated sequence-structure database, called IADE (Integrated ASTRAL-DSSP-EMBL), incorporating matching mRNA sequence, amino acid sequence, and protein secondary structural data, is constructed. It includes 648 protein domains. Based on the IADE database, we studied the relation between RNA stem-loop frequencies and protein secondary structure. It was found that the alpha-helices and beta-strands on proteins tend to be preferably "coded" by mRNA stem region, while the coils on proteins tend to be preferably "coded" by mRNA loop region. These tendencies are more obvious if we observe the structural words (SWs). An SW is defined by a four-amino-acid-fragment that shows the pronounced secondary structural (alpha-helix or beta-strand) propensity. It is demonstrated that the deduced correlation between protein and mRNA structure can hardly be explained as the stochastic fluctuation effect. (C) 2003 Wiley Periodicals, Inc.

Cardioviral RNA structure logo analysis: entropy, correlations, and prediction

In recent years, there has been an increased number of sequenced RNAs leading to the development of new RNA databases. Thus, predicting RNA structure from multiple alignments is an important issue to understand its function. Since RNA secondary structures are often conserved in evolution, developing methods to identify covariate sites in an alignment can be essential for discovering structural elements. Structure Logo is a technique established on the basis of entropy and mutual information measured to analyze RNA sequences from an alignment. We proposed an efficient Structure Logo approach to analyze conservations and correlations in a set of Cardioviral RNA sequences. The entropy and mutual information content were measured to examine the conservations and correlations, respectively. The conserved secondary structure motifs were predicted on the basis of the conservation and correlation analyses. Our predictive motifs were similar to the ones observed in the viral RNA structure database, and the correlations between bases also corresponded to the secondary structure in the database.

RNA干扰在抗病毒研究中的应用

国家自然科学基金海外杰出青年基金(30428024)资助

双链RNA病毒诱导的牙鲆胚胎细胞差减cDNA文库的构建

以紫外线灭活的dsRNA病毒草鱼出血病病毒(GCHV)诱导和模拟诱导的牙鲆胚胎细胞为材料,利用抑制性差减杂交(SSH)技术,成功构建了双链RNA病毒诱导的牙鲆胚胎细胞(FEC)差减cDNA文库。以管家基因αtub lin作为差减指标,经检测,该文库差减效率达210倍,表明经病毒诱导后某些差异表达基因也得到了相应倍数的富集。将获得的cDNA片段连接到pGEM T载体,PCR检测显示差减片段在250bp~2 000bp之间。该差减cDNA文库的构建为从分子水平研究牙鲆培养细胞对dsRNA病毒的免疫反应、以及进

草鱼呼肠孤病毒RNA聚合酶基因的表达与产物纯化

草鱼呼肠孤病毒是引起草鱼出血病的主要病原,隶属于呼肠孤病毒科水生呼肠孤病毒属。序列分析表明,GCRV S2 片段长为3 877核苷酸,编码一个分子量为138kDa 的蛋白VP2,具有RNA聚合酶性质。为进一步了解该病毒 RNA聚合酶特性,本研究在对GCRV RNA聚合酶基因(GCRV-RdRp)保守区(约1.5kb)重组质粒pR/RRp高效表达的基础上,分别构建了编码GCRV RNA聚合酶保守区N端与C端部分基因的 pR/RRpN及pR/RRpC重组表达载体,并在原核细胞中获得成功表达。筛选的重组表达菌株

草鱼呼肠孤病毒RNA聚合酶基因功能区在原核细胞中的表达

国家自然科学基金资助项目 ( 30 170 730 )

RNA剪接和剪接调控

ＲＮＡ剪接和剪接调控桂建芳（中国科学院水生生物研究所武汉４３００７２）基因是遗传的功能单位，蛋白是由基因表达的行使生理功能的主体。７０年代以前，人们对基因和蛋白的了解主要是来自于对细菌研究的认识，认为基因、ｍＲＮＡ和蛋白之间是一种线性关系，即基因上的...

鲤鱼白肌中RNA/DNA值与其生长的关系

鲤鱼白肌中核糖核酸与脱氧核糖核酸的比值(RNA/DNA)可作为种群生长的生理指标,并用此指标预测和评价生态环境、饲养条件的优劣对其生长可能产生的影响。鱼体白肌中RNA/DAN值与其生长率呈正相关(r=0.8994);鱼体增重率和RNA/DNA值的季节变动规律基本一致,都是在9-10月最高,4月次之,11-12月最低。鲤鱼生长良好时,RNA/DNA值大于2.0;反之,低于2.0;Hg~(2+)浓度达到0.005mg/l时,才对鲤鱼的生长产生显著影响,并在RNA/DNA值上显示出来。用RNA/DNA值评价鱼的

Reconsideration of Phylogenetic Relationships of the Subclass Peritrichia (Ciliophora, Oligohymenophorea) Based on Small Subunit Ribosomal RNA Gene Sequences, with the Establishment of a New Subclass Mobilia Kahl, 1933

Based on its characteristic oral apparatus, the ciliate subclass Peritrichia has long been recognized as a monophyletic assemblage composed of the orders Mobilida and Sessilida. Following the application of molecular methods, the monophyly of Peritrichia has recently been questioned. We investigated the phylogenetic relationships of the peritrichous ciliates based on four further complete small subunit ribosomal RNA sequences of mobilids, namely Urceolaria urechi, Trichodina meretricis, Trichodina sinonovaculae, and Trichodina ruditapicis. In all phylogenetic trees, the mobilids never clustered with the sessilids, but instead formed a monophyletic assemblage related to the peniculines. By contrast, the sessilids formed a sister clade with the hymenostomes at a terminal position within the Oligohymenophorea. We therefore formally separate the mobilids from the sessilids (Peritrichia sensu stricto) and establish a new subclass, Mobilia Kahl, 1933, which contains the order Mobilida Kahl, 1933. We argue that the oral apparatus in the mobilians and sessilid peritrichs is a homoplasy, probably due to convergent evolution driven by their similar life-styles and feeding strategies. Morphologically, the mobilians are distinguished from all other oligohymenophoreans by the presence of the adhesive disc, this character being a synapomorphy for the Mobilia.

Transcriptome of embryonic and neonatal mouse cortex by high-throughput RNA sequencing

Brain structure and function experience dramatic changes from embryonic to postnatal development. Microarray analyses have detected differential gene expression at different stages and in disease models, but gene expression information during early brain development is limited. We have generated >27 million reads to identify mRNAs from the mouse cortex for>16,000 genes at either embryonic day 18 (E18) or postnatal day 7 (P7), a period of significant synapto-genesis for neural circuit formation. In addition, we devised strategies to detect alternative splice forms and uncovered more splice variants. We observed differential expression of 3,758 genes between the 2 stages, many with known functions or predicted to be important for neural development. Neurogenesis-related genes, such as those encoding Sox4, Sox11, and zinc-finger proteins, were more highly expressed at E18 than at P7. In contrast, the genes encoding synaptic proteins such as synaptotagmin, complexin 2, and syntaxin were up-regulated from E18 to P7. We also found that several neurological disorder-related genes were highly expressed at E18. Our transcriptome analysis may serve as a blueprint for gene expression pattern and provide functional clues of previously unknown genes and disease-related genes during early brain development.

Origin and evolution of the RIG-I like RNA helicase gene family

Background: The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria. Results: The DExD/H containing RNA helicases including RIG-I, MDA5 and LGP2 were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. The gene synteny of MDA5 and LGP2 is well conserved among vertebrates whilst conservation of the gene synteny of RIG-I is less apparent. Invertebrate homologues had a closer phylogenetic relationship with the vertebrate RIG-Is than the MDA5/LGP2 molecules, suggesting the RIG-I homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. Our data suggest that the RIG-I like helicases possibly originated from three distinct genes coding for the core domains including the HELICc, CARD and ATP dependent DExD/H domains through gene fusion and gene/domain duplication. Furthermore, presence of domains similar to a prokaryotic DNA restriction enzyme III domain (Res III), and a zinc finger domain of transcription factor (TF) IIS have been detected by bioinformatic analysis. Conclusion: The RIG-I/MDA5 viral surveillance system is conserved in vertebrates. The RIG-I like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. Diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral PAMPs.

Isolation and characterization of Argonaute 2: A key gene of the RNA interference pathway in the rare minnow, Gobiocypris rarus

Argonaute 2 gene plays a pivotal role in RNAi in many species. Herein is the first report of the cloning and characterization of Argonaute 2 gene in fish. The full-length cDNA of Gobiocypris rarus Argonaute 2 (GrAgo2) consisted of 3073 nucleotides encoding 869 amino acid residues with a calculated molecular weight of 98.499 kDa and an estimated isoelectric point of 9.18. Analysis of the deduced amino acid sequence showed the presence of two signature domains, PAZ and Piwi. RT-PCR analysis indicated that GrAgo2 mRNA expression could be detected in widespread tissues. After infection with grass carp reovirus, GrAgo2 expression was up-regulated from 12 h post-injection (p < 0.05) and returned to control levels at 48 h post-injection (p > 0.05). These data imply that GrAgo2 is involved in antiviral defense in rare minnow. (C) 2008 Published by Elsevier Ltd.

Hybrid cytomegalovirus-U6 promoter-based plasmid vectors improve efficiency of RNA interference in zebrafish

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.

Functional domains and the antiviral effect of the double-stranded RNA-dependent protein kinase PKR from Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase PKR is thought to mediate a conserved antiviral pathway by inhibiting viral protein synthesis via the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2 alpha). However, little is known about the data related to the lower vertebrates, including fish. Recently, the identification of PKR-like, or PKZ, has addressed the question of whether there is an orthologous PKR in fish. Here, we identify the first fish PKR gene from the Japanese flounder Paralichthys olivaceus (PoPKR). PoPKR encodes a protein that shows a conserved structure that is characteristic of mammalian PKRs, having both the N-terminal region for dsRNA binding and the C-terminal region for the inhibition of protein translation. The catalytic activity of PoPKR is further evidence that it is required for protein translation inhibition in vitro. PoPKR is constitutively transcribed at low levels and is highly induced after virus infection. Strikingly, PoPKR overexpression increases eIF2 alpha phosphorylation and inhibits the replication of Scophthalmus maximus rhabdovirus (SMRV) in flounder embryonic cells, whereas phosphorylation and antiviral effects are impaired in transfected cells expressing the catalytically inactive PKR-K421R variant, indicating that PoPKR inhibits virus replication by phosphorylating substrate eIF2 alpha. The interaction between PoPKR and eIF2 alpha is demonstrated by coimmunoprecipitation assays, and the transfection of PoPKR-specific short interfering RNA further reveals that the enhanced eIF2 alpha phosphorylation is catalyzed by PoPKR during SMRV infection. The current data provide significant evidence for the existence of a PKR-mediated antiviral pathway in fish and reveal considerable conservation in the functional domains and the antiviral effect of PKR proteins between fish and mammals.