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

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.

Variation patterns of the mitochondrial 16S rRNA gene with secondary structure constraints and their application to phylogeny of cyprinine fishes (Teleostei : Cypriniformes)

The mitochondrial 16S ribosomal RNA (rRNA) gene sequences from 93 cyprinid fishes were examined to reconstruct the phylogenetic relationships within the diverse and economically important subfamily Cyprininae. Within the subfamily a biased nucleotide composition (A > T, C > G) was observed in the loop regions of the gene, and in stem regions apparent selective pressures of base pairing showed a bias in favor of G over C and T over A. The bias may be associated with transition-transversion bias. Rates of nucleotide substitution were lower in stems than in loops. Analysis of compensatory substitutions across these taxa demonstrates 68% covariation in the gene and a logical weighting factor to account for dependence in mutations for phylogenetic inference should be 0.66. Comparisons of varied stem-loop weighting schemes indicate that the down-weightings for stem regions could improve the phylogenetic analysis and the degree of non-independence of stem substitutions was not as important as expected. Bayesian inference under four models of nucleotide substitution indicated that likelihood-based phylogenetic analyses were more effective in improving the phylogenetic performance than was weighted parsimony analysis. In Bayesian analyses, the resolution of phylogenies under the 16-state models for paired regions, incorporating GTR + G + I models for unpaired regions was better than those under other models. The subfamily Cyprininae was resolved as a monophyletic group, as well as tribe Labein and several genera. However, the monophyly of the currently recognized tribes, such as Schizothoracin, Barbin, Cyprinion + Onychostoma lineages, and some genera was rejected. Furthermore, comparisons of the parsimony and Bayesian analyses and results of variable length bootstrap analysis indicates that the mitochondrial 16S rRNA gene should contain important character variation to recover well-supported phylogeny of cyprinid taxa whose divergences occurred within the recent 8 MY, but could not provide resolution power for deep phylogenies spanning 10-19 MYA. (c) 2008 Published by Elsevier Inc.

Molecular phylogenetics of the family Cyprinidae (Actinopterygii : Cypriniformes) as evidenced by sequence variation in the first intron of S7 ribosomal protein-coding gene: Further evidence from a nuclear gene of the systematic chaos in the family

The family Cyprinidae is the largest freshwater fish group in the world, including over 200 genera and 2100 species. The phylogenetic relationships of major clades within this family are simply poorly understood, largely because of the overwhelming diversity of the group; however, several investigators have advanced different hypotheses of relationships that pre- and post-date the use of shared-derived characters as advocated through phylogenetic systematics. As expected, most previous investigations used morphological characters. Recently, mitochondrial DNA (mtDNA) sequences and combined morphological and mtDNA investigations have been used to explore and advance our understanding of species relationships and test monophyletic groupings. Limitations of these studies include limited taxon sampling and a strict reliance upon maternally inherited mtDNA variation. The present study is the first endeavor to recover the phylogenetic relationships of the 12 previously recognized monophyletic subfamilies within the Cyprinidae using newly sequenced nuclear DNA (nDNA) for over 50 species representing members of the different previously hypothesized subfamily and family groupings within the Cyprinidae and from other cypriniform families as outgroup taxa. Hypothesized phylogenetic relationships are constructed using maximum parsimony and Basyesian analyses of 1042 sites, of which 971 sites were variable and 790 were phylogenetically informative. Using other appropriate cypriniform taxa of the families Catostomidae (Myxocyprinus asiaticus), Gyrinocheilidae (Gyrinocheilus aymonieri), and Balitoridae (Nemacheilus sp. and Beaufortia kweichotvensis) as outgroups, the Cyprinidae is resolved as a monophyletic group. Within the family the genera Raiamas, Barilius, Danio, and Rasbora, representing many of the tropical cyprinids, represent basal members of the family. All other species can be classified into variably supported and resolved monophyletic lineages, depending upon analysis, that are consistent with or correspond to Barbini and Leuciscini. The Barbini includes taxa traditionally aligned with the subfamily Cyprininae sensu previous morphological revisionary studies by Howes (Barbinae, Labeoninae, Cyprininae and Schizothoracinae). The Leuciscini includes six other subfamilies that are mainly divided into three separate lineages. The relationships among genera and subfamilies are discussed as well as the possible origins of major lineages. (c) 2008 Published by Elsevier Inc.

The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo

The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. In the present study, we used a cytomegalovirus (CMV) promoter-driven DNA template approach to induce short hairpin RNA (shRNA) triggered RNAi to block exogenous Enhanced Green Fluorescent Protein (EGFP) and endogenous No Tail (NTL) gene expressions. We constructed three plasmids, pCMV-EGFP-CMV-shGFP-SV40, pCMV-EGFP-CMV-shNTL-SV40, and pCMV-EGFP-CMV-shScrambled-SV40, each containing a CMV promoter driving an EGFP reporter cDNA and DNA coding for one shRNA under the control of another CMV promoter. The three shRNA-generating plasmids and pCMV-EGFP control plasmid were introduced into zebrafish embryos by microinjection. Samples were collected at 48 h after injection. Results were evaluated by phenotype observation and real-time fluorescent quantitative reverse-transcription polymerase chain reaction (Q-PCR). The shGFP-generating plasmid significantly inhibited the EGFP expression viewed under fluorescent microscope and reduced by 70.05 +/- 1.26% of exogenous EGFP gene mRNA levels compared with controls by Q-PCR. The shRNA targeting endogenous NTL gene resulted in obvious NTL phenotype of 30 +/- 4% and decreased the level of their corresponding mRNAs up to 54.52 +/- 2.05% compared with nontargeting control shRNA. These data proved the feasibility of the CMV promoter-driven shRNA expression technique to be used to inhibit exogenous and endogenous gene expressions in zebrafish in vivo.

Alternative cold response modes in Chlorella (Chlorophyta, Trebouxiophyceae) from Antarctica

Chlorella was known to show enhanced antifreeze capability after cold hardening. We isolated Chlorella strains NJ-7 and NJ-18, which display alternative cold response modes from rock surfaces in Antarctica. On the basis of 18S ribosomal (rRNA) sequences, NJ-7 is an Antarctic type of Chlorella vulgaris; NJ-18 is also a 'true' Chlorella species but differs from any previously reported species in structure. NJ-7 partially retained the enhancing effects of low temperature cultivation on freeze tolerance, which correlates with an increase of C18:3-fatty acid content and up-regulation of two antifreeze protein genes. NJ-18, however, showed stable freeze tolerance regardless of the precultivation temperature. We propose that cold response modes vary widely in Chlorella and that the adaptation of C. vulgaris to Antarctica may serve as a model system for the evolution of antifreeze mechanisms in a single species of photosynthetic microorganism.

Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system

A short-hairpin RNA (shRNA) expression system, based on T7 RNA polymerase (T7RP) directed transcription machinery, has been developed and used to generate a knock down effect in zebrafish embryos by targeting green fluorescent protein (gfp) and no tail (ntl) mRNA. The vector pCMVT7R harboring T7RP driven by CMV promoter was introduced into zebrafish embryos and the germline transmitted transgenic individuals were screened out for subsequent RNAi application. The shRNA transcription vectors pT7shRNA were constructed and validated by in vivo transcription assay. When pT7shGFP vector was injected into the transgenic embryos stably expressing T7RP, gfp relative expression level showed a decrease of 68% by analysis of fluorescence real time RT-PCR. As a control, injection of chemical synthesized siRNA resulted in expression level of 40% lower than the control when the injection dose was as high as 2 mu g/mu l. More importantly, injection of pT7shNTL vector in zebrafish embryos expressing T7RP led to partial absence of endogenous ntl transcripts in 30% of the injected embryos when detected by whole mount in situ hybridization. Herein, the T7 transcription system could be used to drive the expression of shRNA in zebrafish embryos and result in gene knock down effect, suggesting a potential role for its application in RNAi studies in zebrafish embryos.

Phylogeny of Oedogoniales (Chlorophyceae, Chlorophyta) inferred from 18S rDNA sequences with emphasis on the relationships in the genus Oedogonium based on ITS-2 sequences

The phylogeny of Oedogoniales was investigated by using nuclear 18S rDNA sequences. Results showed that the genus Oedocladium, as a separated clade, was clustered within the clade of Oedogonium; whereas the genus Bulbochaete was in a comparatively divergent position to the other two genera. The relationship among the species of Oedogonium was discussed, focusing on ITS-2 phylogeny analyzed combining with some morphological characteristics. Our results showed that all the dioecious nannandrous taxa involved in this study were resolved into one clade, while all the monocious taxa were clustered into another clade as a sister group to the former. The report also suggests that the dioecious macrandrous taxa form a paraphyly and could be more basally situated than the dioecious nannandrous and the monoecious taxa by means of molecular phylogeny and morphotype investigations.

Redescription of Trypanosoma siniperca Chang 1964 from freshwater fish of China based on morphological and molecular data

During the parasite fauna investigation within 2005, the freshwater fish trypanosome Trypanosoma siniperca Chang 1964 was isolated from the blood of Mandarin carp (Siniperca chuatsi) from Niushan Lake, Hubei Province, central China. Blood trypomastigotes were observed only, and the density of infection was low. Light microscopy examinations of this material made it possible to study in detail the morphology of this parasite and redescribe it according to current standards. T. siniperca is characterized also on the molecular level using the sequences of SSU rRNA gene. Phylogenetic analyses based on these sequences allowed clearer phylogenetic relationships to be established with other fish trypanosomes sequenced to date.

The complete mitochondrial genome of the helmet catfish Cranoglanis bouderius (Silurifonnes : Cranoglanididae) and the phylogeny of otophysan fishes

The complete sequence of the 16,539 nucleotide mitochondrial genome from the single species of the catfish family Cranoglanididae, the helmet catfish Cranoglanis bouderius, was determined using the long and accurate polymerase chain reaction (LA PCR) method. The nucleotide sequences of C. bouderius mitochondrial DNA have been compared with those of three other catfish species in the same order. The contents of the C. bouderius mitochondrial genome are 13 protein-coding genes, two ribosomal RNA and 22 transfer RNA genes, and a non-coding control region, the gene order of which is identical to that observed in most other vertebrates. Phylogenetic analyses for 13 otophysan fishes were performed using Bayesian method based on the concatenated mtDNA protein-coding gene sequence and the individual protein-coding gene sequence data set. The competing otophysan topologies were then tested by using the approximately unbiased test, the Kishino-Hasegawa test, and the Shimodaira-Hasegawa test. The results show that the grouping ((((Characifonnes, Gymnotiformes), Siluriformes), Cyprinifionnes), outgroup) is the most likely but there is no significant difference between this one and the other alternative hypotheses. In addition, the phylogenetic placement of the family Cranoglanididae among siluriform families was also discussed. (c) 2006 Elsevier B.V. All rights reserved.