Effects of exogenous double-stranded RNA on the basonuclin gene expression in mouse oocytes

In plants and less-advanced animal species, such as C.elegans, introduction of exogenous double-stranded RNA (dsRNA) into cells would trigger degradation of the mRNA with homologous sequence and interfere with the endogenous gene expression. It might represent an ancient anti-virus response which could prevent the mutation in the genome that was caused by virus infection or mobile DNA elements insertion. This phenomenon was named RNA interference, or RNAi. In this study, RNAi was used to investigate the function of basonuclin gene during oogenesis. Microinjection of dsRNA directed towards basonuclin into mouse germinal-vesicle-intact (GV) oocytes brought down the abundance of the cognate mRNA effectively in a time- and concentration-dependent manner. This reduction effect was sequence-specific and showed no negative effect on other non-homologous gene expression in oocytes, which indicated that dsRNA can recognize and cause the degradation of the transcriptional products of endogenous basonuclin gene in a sequence-specific manner. Immunofluorescence results showed that RNAi could reduce the concentration of basonuclin protein to some extent, but the effect was less efficient than the dsRNA targeting towards tPA and cMos which was also expressed in oocytes. This result might be due to the long half life of basonuclin protein in oocytes and the short reaction time which was posed by the limited life span of GV oocytes cultured in vitro. In summary, dsRNA could inhibit the expression of the cognate gene in oocytes at both mRNA and protein levels. The effect was similar to Knock-out technique which was based on homologous recombination. Furthermore, hairpin-style dsRNA targeting basonuclin gene could be produced by transcription from a recombinant plasmid and worked efficiently to deplete the cognate mRNA in oocytes. This finding offered a new way to study the function of basonuclin in the early stage of oogenesis by infection of primordial oocytes with the plasmid expressing hairpin-style basonuclin dsRNA.

Glucocorticoid receptor and protein/RNA synthesis-dependent mechanisms underlie the control of synaptic plasticity by stress

Learning and memory are exquisitely sensitive to behavioral stress, but the underlying mechanisms are still poorly understood. Because activity-dependent persistent changes in synaptic strength are believed to mediate memory processes in brain areas such as the hippocampus we have examined the means by which stress affects synaptic plasticity in the CA1 region of the hippocampus of anesthetized rats, Inescapable behavioral stress (placement on an elevated platform for 30 min) switched the direction of plasticity, favoring low frequency stimulation-induced decreases in synaptic transmission (long-term depression, LTD), and opposing the induction of long-term potentiation by high frequency stimulation, We have discovered that glucocorticoid receptor activation mediates these effects of stress on LTD and longterm potentiation in a protein synthesis-dependent manner because they were prevented by the glucocorticoid receptor antagonist RU 38486 and the protein synthesis inhibitor emetine. Consistent with this, the ability of exogenously applied corticosterone in non-stressed rats to mimic the effects of stress on synaptic plasticity was also blocked by these agents, The enablement of low frequency stimulation-induced LTD by both stress and exogenous corticosterone was also blocked by the transcription inhibitor actinomycin D, Thus, naturally occurring synaptic plasticity is liable to be reversed in stressful situations via glucocorticoid receptor activation and mechanisms dependent on the synthesis of new protein and RNA, This indicates that the modulation of hippocampus-mediated learning by acute inescapable stress requires glucocorticoid receptor-dependent initiation of transcription and translation.

RNA 的历史与未来

核酶的发现使得人们有理由相信生命起源于RNA ,通过试管演化实验获得的各种各样的催化性RNA 更使人们 对地球历史早期的RNA 世界有了越来越多的了解。同时,随着RNA 结构和功能上非凡的多样性的日益被揭示,RNA 在未来的临床应用研究中所具有的巨大潜力也正逐渐显现出来。

稳定的RNA 发夹结构在人类88 个mRNA 编码区中的分布

　以UNCG, GNRA , CUU G (N = A , U , C 或G; R = G或A) 为端环能够形成稳定的、保 守的发夹结构. 它们具有特殊的结构特征, 并在体内发挥着重要的生物学功能. 这些稳定的发夹 广泛分布于体内rRNA , 催化RNA 和非编码mRNA 中. 但对人类88 个编码区mRNA 二级结构的 研究当中, 却没有发现C(UUCG) G发夹. 而且, 与rRNA 不同, 这些编码区mRNA 四环序列的 分布没有明显的偏好性.

RNA A_6膨胀环的动力学特征

通过对RNA A6膨胀环在水溶液中的动力学模拟发现,在A6膨胀环中,环区构象主要以非堆积构象为主,环区具有较大的构象柔性,膨胀环区链的构象波动对已形成的RNA分子弯折的影响不大,弯折角只是在小范围内变动,提示作为全局结构,带大尺寸膨胀环的RNA分子仍然具有一定的刚性,柔性主要表现在膨胀环区域.

Additional angles to calculate the deformation of RNA helices induced by bulge loops

Bulges are common features of folded RNA structures. The RNA axial kinking caused by bulges has been confirmed by many experiments. Usually, a kinking angle zeta and a bending angle theta are used to describe the kinking and twisting of RNA molecules containing bulges. Here, we present two additional angles (twist angle zeta(1), twist angle zeta(2)) to describe the deformation of RNA helices induced by bulge loops because only two angles (a kinking angle zeta and a bending angle theta) are not enough to define the deformation of RNA induced by bulges. (C) 2002 Elsevier Science B.V. All rights reserved.

Dynamics of RNA with A6 bulge loop

In order to understand the behavior of RNAs with large bulges In solution, molecular dynamics was performed on the RNA molecule in water with A6 bulge. The result of simulation showed that nonstacked conformation Is the main conformation in large bulges, and the backbone of large bulge is of great conformational flexibility, but bulges-induced bends are relatively rigid. The fluctuation in bulge has little influence on the bend angle of RNAs.

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.

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