A novel small stable RNA, 6Sa RNA,from the cyanobacterium Synechococcus sp. strain PCC6301

We isolated a novel RNA species from the unicellular cyanobacterium Synechococcus PCC6301 and determined its gene sequence. This novel RNA was termed 6Sa RNA from its length (185 nt). Cross-hybridization of 6Sa RNA to other related microorganisms suggests that its existence is restricted to the Synechococcus genus or related organisms. A high level of accumulation of this RNA was observed by Northern analysis, indicating that 6Sa RNA is stable in cells. Computer-aided prediction of the 6Sa RNA secondary structure also supports its stability.     

Location of a gene (ssrA) for a small, stable RNA (10Sa RNA) in the Escherichia coli chromosome.

The gene for 10Sa RNA, which is a major small, stable RNA in Escherichia coli, is a unique gene in the E. coli chromosome. The 10Sa RNA gene (ssrA) has been located between 2,760 and 2,761 kilobases on the E. coli genome.     

Characterization of 6S RNA in the Lyme disease spirochete

6S RNA binds to RNA polymerase and regulates gene expression, contributing to bacterial adaptation to environmental stresses. In this study, we examined the role of 6S RNA in murine infectivity and tick persistence of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi. B. burgdorferi 6S RNA (Bb6S RNA) binds to RNA polymerase, is expressed independent of growth phase or nutrient stress in culture, and is processed by RNase Y. We found that rny (bb0504), the gene encoding RNase Y, is essential for B. burgdorferi growth, while ssrS, the gene encoding 6S RNA, is not essential, indicating a broader role for RNase Y activity in the spirochete. Bb6S RNA regulates expression of the ospC and dbpA genes encoding outer surface protein C and decorin binding protein A, respectively, which are lipoproteins important for host infection. The highest levels of Bb6S RNA are found when the spirochete resides in unfed nymphs. ssrS mutants lacking Bb6S RNA were compromised for infectivity by needle inoculation, but injected mice seroconverted, indicating an ability to activate the adaptive immune response. ssrS mutants were successfully acquired by larval ticks and persisted through fed nymphs. Bb6S RNA is one of the first regulatory RNAs identified in B. burgdorferi that controls the expression of lipoproteins involved in host infectivity.     

Chitosan nanoparticles help double-stranded RNA escape from endosomes and improve RNA interference in the fall armyworm,Spodoptera frugiperda

RNA interference (RNAi) is a promising technology for the development of next-generation insect pest control products. Though RNAi is efficient and systemic in coleopteran insects, it is inefficient and variable in lepidopteron insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda by conjugating double-stranded RNA (dsRNA) with biodegradable chitosan (Chi). dsRNA conjugated with chitosan was protected from degradation by endonucleases present in Sf9 cell-conditioned medium, hemolymph, and midgut lumen contents collected from the FAW larvae. Chi–dsRNA complexes showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing chitosan formulated dsRNA in Sf9 cells and the tissues induced a significant knockdown of endogenous genes. Chi–dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation, and mortality. Processing of dsRNA into small interfering RNA was detected with chitosan-conjugated ³²P-UTP-labeled ds green fluorescent protein in Sf9 cells and FAW larval tissues. Overall, these data suggest that dsRNA conjugated with chitosan helps dsRNA escape from the endosomes and improves RNAi efficiency in FAW cells and tissues.     

Genome-wide bioinformatic prediction and experimental evaluation of potential RNA thermometers

Only recently, the fundamental role of regulatory RNAs in prokaryotes and eukaryotes has been appreciated. We developed a pipeline from bioinformatic prediction to experimental validation of new RNA thermometers. Known RNA thermometers are located in the 5′-untranslated region of certain heat shock or virulence genes and control translation by temperature-dependent base pairing of the ribosome binding site. We established the searchable database RNA-SURIBA (Structures of Untranslated Regions In BActeria). A structure-based search pattern reliably recognizes known RNA thermometers and predicts related structures upstream of annotated genes in complete genome sequences. The known ROSE₁ (Repression Of heat Shock gene Expression) thermometer and several other functional ROSE-like elements were correctly predicted. For further investigation, we chose a new candidate upstream of the phage shock gene D (pspD) in the pspABCDE operon of E. coli. We established a new reporter gene system that measures translational control at heat shock temperatures and we demonstrated that the upstream region of pspD does not confer temperature control to the phage shock gene. However, translational efficiency was modulated by a point mutation stabilizing the predicted hairpin. Testing other candidates by this structure prediction and validation process will lead to new insights into the requirements for biologically active RNA thermometers. The database is available on . Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

NRSF慢病毒干涉载体的构建及功能初步检测

为研究神经元限制性沉默因子(NRSF)负调控神经元及胰岛细胞中神经特异性基因的表达,拟通过RNAi的方法降低NRSF表达以进一步观察其调控的下游基因的表达情况.构建含人胰岛素启动子-荧光素酶(HIP-LUC)的pcDNA3.1报告载体.通过RNAi序列设计软件进行NRSF干涉片段及脱靶对照片段的设计,然后构建慢病毒干涉载体.包装产生慢病毒干涉毒液,用其感染HeLa细胞获得稳定干涉NRSF的细胞株,利用RT-PCR、实时定量PCR、蛋白质免疫印迹、免疫荧光染色等方法检测NRSF的干涉效果及其下游基因的表达情况,观察干涉NRSF后胰岛素启动子报告载体荧光素酶活性的变化.构建具有NRSF干涉效果的慢病毒干涉载体成功,并获得了稳定干涉NRSF及脱靶对照的HeLa细胞株,RT-PCR及实时定量PCR检测结果表明,NRSF干涉片段的干涉效率为56%(n=6,P<0.01),蛋白质免疫印迹、免疫荧光染色方法检测证实干涉后NRSF蛋白表达水平明显降低.RT-PCR实验表明干涉NRSF后下游基因开始表达,荧光素酶活性分析表明,干涉NRSF后胰岛素启动子活性增强了2.4倍(n=3,P<0.01).上述结果表明,成功构建NRSF的慢病毒干涉载体并获得稳定干涉NRSF的HeLa细胞株,干涉NRSF后,其下游基因特别是胰岛素基因开始表达.这一研究工作有助于我们进一步了解NRSF在胰岛细胞发育分化中的调控作用.     

Characterization of the RNA processing enzyme RNase III from wild type and overexpressing Escherichia coli cells in processing natural RNA substrates.

1. A precursor to small stable RNA, 10Sa RNA, accumulates in large amounts in a temperature sensitive RNase E mutant at non-permissive temperatures, and somewhat in an rnc (RNase III-) mutant, but not in an RNase P- mutant (rnp) or wild type E. coli cells. 2. Since p10Sa RNA was not processed by purified RNase E and III in customary assay conditions, we purified p10Sa RNA processing activity about 700-fold from wild type E. coli cells. 3. Processing of p10Sa RNA by this enzyme shows an absolute requirement for a divalent cation with a strong preference for Mn2+ over Mg2+. Other divalent cations could not replace Mn2+. 4. Monovalent cations (NH+4, Na+, K+) at a concentration of 20 mM stimulated the processing of p10Sa RNA and a temperature of 37 degrees C and pH range of 6.8-8.2 were found to be optimal. 5. The enzyme retained half of its p10Sa RNA processing activity after 30 min incubation at 50 degrees C. 6. Further characterization of this activity indicated that it is RNase III. 7. To further confirm that the p10Sa RNA processing activity is RNase III, we overexpressed the RNase III gene in an E. coli cells that lacks RNase III activity (rnc mutant) and RNase III was purified using one affinity column, agarose.poly(I).poly(C). 8. This RNase III preparation processed p10Sa RNA in a similar way as observed using the p10Sa RNA processing activity purified from wild type E. coli cells, confirming that the first step of p10Sa RNA processing is carried out by RNase III.     

RNA干扰技术在果蝇中的应用

RNA干扰是双链RNA特异诱导的转录后期基因沉默.该技术随着不断完善而越来越被广泛地运用于果蝇的功能基因组研究上,双链RNA已经成为果蝇中功能基因的一个十分有效的抑制子,势必使RNA干扰技术成为研究果蝇体内基因功能的强有力的反向遗传学研究技术.     

RNase P RNA ofMycoplasma capricolum

There are at least six small stable RNAs inMycoplasma capricolum cells besides tRNAs and rRNAs. One of them, MCS5 RNA, is a homolog of RNase P RNA. The predicted secondary structure of this RNA is essentially the same as that of other eubacterial RNase P RNAs. MCS5 RNA is more similar to the RNase P RNA ofB. subtilis than to that ofE. coli. This is consistent with previous conclusions that mycoplasmas are phylogenetically related to the low G+C Gram-positive bacterial group. The major substrates for MCS5 RNA must be the precursors of tRNAs. The precursor of MCS6 RNA, which is a homolog of theE. coli 10Sa RNA, may also be a substrate for the MCS5 RNA because this RNA has a tRNA-like structure at its 5 and 3 ends.     

Chimeric RNA/DNA oligonucleotide-directed gene targeting in rice

Site-specific mutagenesis in a rice genome was obtained by introducing chimeric RNA/DNA oligonucleotides (COs) by means of particle bombardment. Three COs were designed to target the independent codons for Pro-171, Trp-548 and Ser-627 of the endogenous rice acetolactate synthase (ALS) gene so it would confer resistance to ALS-inhibiting herbicides. Sequencing of the ALS gene of herbicide-resistant plants demonstrated that the ALS sequence was modified in a site-specific fashion. The efficiency of gene conversion mediated by COs was estimated to be 1×10^-4. These results demonstrate that CO-directed gene targeting is feasible in rice.Abbreviations ALS Acetolactate synthase - BS Bispyribac-sodium - Cf Chlorsulfuron - CO Chimeric RNA/DNA oligonucleotide Communicated by H. Uchimiya     

A Transient RNA Interference Assay System Using Arabidopsis Protoplasts

Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana.     

Lipids help double-stranded RNA in endosomal escape and improve RNA interference in the fall armyworm,Spodoptera frugiperda

RNA interference (RNAi) is a valuable method for understanding the gene function and holds great potential for insect pest management. While RNAi is efficient and systemic in coleopteran insects, RNAi is inefficient in lepidopteran insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda cells by formulating dsRNA with Cellfectin II (CFII) transfection reagent. The CFII formulated dsRNA was protected from degradation by endonucleases present in Sf9 cells conditioned medium, hemolymph and midgut lumen contents collected from the FAW larvae. Lipid formulated dsRNA also showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing Sf9 cells and tissues to CFII formulated dsRNA caused a significant knockdown of endogenous genes. CFII formulated dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation and mortality. Processing of dsRNA into siRNA was detected in Sf9 cells and Spodoptera frugiperda larvae treated with CFII conjugated ³²P-UTP labeled dsGFP. Overall, the present study concluded that delivering dsRNA formulated with CFII transfection reagent helps dsRNA escapes from the endosomal accumulation and improved RNAi efficiency in the FAW cells and tissues.     

Structure-function analysis in nuclear RNase P RNA

Eukaryotic ribonuclease P (RNase P) enzymes require both RNA and protein subunits for activityin vivo andin vitro. We have undertaken an analysis of the complex RNA subunit of the nuclear holoenzyme in an effort to understand its structure and its similarities to and differences from the bacterial ribozymes. Phylogenetic analysis, structure-sensitive RNA footprinting, and directed mutagenesis reveal conserved secondary and tertiary structures with both strong similarities to the bacterial consensus and distinctive features. The effects of mutations in the most highly conserved positions are being used to dissect the functions of individual subdomains.Abbreviations RPRI ribonucleaseP ribonucleoprotein 1 gene fromSaccharomyces cerevisiae - Pu purine ribonucleoside