Sequence-non-specific effects of RNA interference triggers and microRNA regulators

RNA reagents of diverse lengths and structures, unmodified or containing various chemical modifications are powerful tools of RNA interference and microRNA technologies. These reagents which are either delivered to cells using appropriate carriers or are expressed in cells from suitable vectors often cause unintended sequence-non-specific immune responses besides triggering intended sequence-specific silencing effects. This article reviews the present state of knowledge regarding the cellular sensors of foreign RNA, the signaling pathways these sensors mobilize and shows which specific features of the RNA reagents set the responsive systems on alert. The representative examples of toxic effects caused in the investigated cell lines and tissues by the RNAs of specific types and structures are collected and may be instructive for further studies of sequence-non-specific responses to foreign RNA in human cells.     

Expression of microRNAs and their precursors in synaptic fractions of adult mouse forebrain

We have characterized the expression of microRNAs and selected microRNA precursors within several synaptic fractions of adult mouse forebrain, including synaptoneurosomes, synaptosomes and isolated post-synaptic densities (PSDs), using methods of microRNA microarray, real time qRT-PCR, Northern blotting and immunopurification using anti-PSD95 antibody. The majority of brain microRNAs (especially microRNAs known to be expressed in pyramidal neurons) are detectably expressed in synaptic fractions, and a subset of microRNAs is significantly enriched in synaptic fractions relative to total forebrain homogenate. MicroRNA precursors are also detectable in synaptic fractions at levels that are comparable to whole tissue. Whereas mature microRNAs are predominantly associated with soluble components of the synaptic fractions, microRNA precursors are predominantly associated with PSDs. For seven microRNAs examined, there was a significant correlation between the relative synaptic enrichment of the precursor and the relative synaptic enrichment of the corresponding mature microRNA. These findings support the proposal that microRNAs are formed, at least in part, via processing of microRNA precursors locally within dendritic spines. Dicer is expressed in PSDs but is enzymatically inactive until conditions that activate calpain cause its liberation; thus, we propose that synaptic stimulation may lead to local processing of microRNA precursors in proximity to the synapse.     

Overexpression of exportin 5 enhances RNA interference mediated by short hairpin RNAs and microRNAs

Plasmids or viral vectors that express short hairpin RNAs (shRNAs) have emerged as important tools for the stable inhibition of specific genes by RNA interference. shRNAs are structural and functional homologs of pre-microRNAs, intermediates in the production of endogenously encoded microRNAs (miRNAs). Therefore, overexpressed shRNAs could inhibit miRNA function by competing for a limiting level of one or more factors involved in miRNA biogenesis or function. Here, we demonstrate that overexpressed shRNAs can saturate the activity of endogenous Exportin 5, a factor required for nuclear export of both shRNAs and pre-miRNAs. While shRNA overexpression can therefore inhibit miRNA function, simultaneous overexpression of Exportin 5 reverses this effect. Moreover, Exportin 5 overexpression can significantly enhance RNA interference mediated by shRNAs. These data have implications for the future clinical utilization of shRNAs and also provide a simple method to enhance RNA interference by shRNAs in culture.     

癌症相关microRNA与靶基因的生物信息学分析

MicroRNAs(miRNAs)是一类长度约为22nt的内源性非编码RNA,通过与靶基因转录本互补结合调控基因的表达。近年来,研究发现miRNA与癌症发生密切相关,miRNA可以直接充当癌基因或者抑癌基因而影响肿瘤的发生和生长。为更进一步揭示癌症相关miRNA的特征及靶基因的功能,文章通过数据库搜索及文献检索,在人类基因组中发现了475个癌症相关miRNA,系统地比较了癌症相关miRNA与非癌症miRNA以及基因内和基因间区癌症相关miRNA在保守性、SNP位点分布、癌谱及转录调控等特性。研究发现,癌症相关miRNA比非癌症miRNA保守性要强,发生SNP概率比较低,同时发现miRNA所涉及癌症数目与保守性成正相关。基因组定位分析发现,癌症相关miRNA比非癌症miRNA更倾向于成簇存在。进一步对宿主基因、癌症相关miRNA及作用的靶基因与癌症发生进行关联分析,发现一些非癌症miRNA的宿主基因倾向于被癌症miRNA作用。本研究结果为深入理解miRNA与癌症之间的关系,以及进一步为miRNA作为癌症诊断指示物提供理论依据。     

An improved method for Southern DNA and Northern RNA blotting using a Mupid-2 Mini-Gel electrophoresis unit

An improved method for Southern DNA and Northern RNA blotting using the Mupid-2 Mini-Gel System is described. We get sharp and clear bands in Southern and Northern blotting after only 30 min short gel electrophoresis instead of the several hours large gel electrophoresis of conventional methods. The high electrical voltage with a pulse-like current of the Mupid-2 Mini-Gel System also allows reduction of the amount of formaldehyde, a harmful reagent, from the gel running buffer in RNA blotting. This minor modification of DNA and RNA blotting technique enables us to perform the complete experimental procedure more quickly economically in less space, than conventional Southern and Northern blotting, as well as using an extremely small amount of formaldehyde in RNA blotting.     

绵羊FGF5基因的克隆、表达及RNA干扰

根据牛的成纤维细胞内生长因子5(Fibroblast growth factor 5,FGF5)基因cDNA序列设计引物,PCR扩增得到绵羊FGF5基因cDNA的开放阅读框序列,并比较和其他6种高等哺乳动物的序列同源性;同时研究该基因在绵羊多种组织的表达情况,以及研究以细胞模型RNA干扰下的表达情况。结果表明,绵羊FGF5基因ORF全长为813 bp,编码270个氨基酸,分子量约为29.58 kDa,理论等电点10.59。绵羊FGF5基因cDNA序列与牛、人、小鼠、大鼠、犬和猫的对应序列同源性高度保守,预测氨基酸序列同源性同样具有高度保守性。RT-PCR分析表明FGF5在绵羊皮肤、小肠、肾脏、心脏、肝脏、脾脏、胰脏和肺中均有表达,皮肤中表达量最高。构建该基因的原核表达载体和RNAi载体,IPTG诱导在大肠杆菌中融合表达获得55 kDa的蛋白条带,设计的RNA干扰片段能显著抑制FGF5基因的表达。文章为进一步阐明绵羊FGF5的功能尤其是在羊毛生长发育中的作用提供了理论和实验基础。     

Functional analysis of cholesterol biosynthesis by RNA interference

Inborn errors of cholesterol biosynthesis caused by dysfunctionality of single enzymes are known to cause severe malformation syndromes like X-linked chondrodysplasia punctata (CDPX2), CHILD syndrome or Smith–Lemli–Opitz-syndrome (SLOS). In this study we established the method of RNA interference (RNAi) for analyzing the molecular mechanisms underlying disrupted cholesterol biosynthesis. For different genes involved in the cholesterol biosynthesis pathway-NAD(P) dependent steroid dehydrogenase-like (NSDHL), 17-beta hydroxysteroid dehydrogenase type 7 (HSD17B7) and emopamil binding protein (EBP)-shRNA sequences were designed and tested for their effectiveness. For a better comparability of the experiments and to avoid different transfection efficiencies, examined shRNA sequences which reached a knock down of at least 80% were stably transfected in a HeLa cell line with a tetracycline-regulated expression (HeLa T-REx). These stable transfected cell lines represent novel tools for the analysis of cholesterol biosynthesis.     

Isomeric analysis of oligomannosidic N-glycans and their dolichol-linked precursors

Oligomannosidic (OM) N-glycans occur as a mixture of isomers, which at early stages of glycosidase trimming also comprise structures with one to three glucose residues. A complementary set of isomers is generated during the biosynthesis of the lipid-linked precursor. Here, we demonstrate the remarkable capacity of liquid chromatography (LC) with porous graphitic carbon and mass spectrometric detection for the determination of OM isomers. Protein-linked N-glycans were released enzymatically from samples with known isomer composition such as kidney bean proteins and ribonuclease B. Lipid-linked oligosaccharides were obtained by a direct mild acid hydrolysis of microsomes thus avoiding biphasic partitioning. A parallel analysis of pyridylaminated glycans by amide-silica and reversed-phase high-performance LC, the application of branch-specific α-mannosidases and work with ALG mutant plants led to the assignment of the relative retention times of the isomers occurring during the degradation of the Glc(3)Man(9)GlcNAc(2) precursor oligosaccharide to Man(5)GlcNAc(2) and beyond. A tightly woven net of evidence supports these assignments. Noteworthy, this isomer assignment happens in the course of a comprehensive analysis of all types of a sample's N-glycans.     

Synaptic enrichment of microRNAs in adult mouse forebrain is related to structural features of their precursors

   

Within mouse forebrain, a subset of microRNAs are significantly enriched in synaptoneurosomes (a synaptic fraction containing pinched-off dendritic spines) and a subset are significantly depleted relative to total forebrain homogenate. Here I show that, as a group, the pre-miR hairpin precursors of synaptically enriched microRNAs exhibit significantly different structural features than those that are non-enriched or depleted. Precursors of synaptically enriched microRNAs tend to have a) shorter uninterrupted double-stranded stem segments, and b) more symmetrical bulges containing a single nucleotide on each side. These structural differences may provide a basis for the differential binding of proteins that mediate dendritic transport of pre-miRs, or that prevent pre-miRs from being prematurely processed into mature miRNAs during the transport process.     

Identification of novel chicken microRNAs and analysis of their genomic organization

MicroRNAs (miRNAs) represent a family of small noncoding RNAs with important regulatory roles in diverse biological processes ranging from cell differentiation to organism development. In chickens, the full set of miRNAs and the expression patterns of miRNAs during development are still poorly understood when compared to the other vertebrates. In this study, we identified 29 novel miRNAs and 140 potential miRNA loci in the chicken genome by combining the experimental and computational analyses. Detailed expression patterns of 49 miRNAs were first characterized by Northern blotting and indicated the cooperativity of the miRNA expression with their function in embryogenesis and organogenesis. Twenty-seven miRNA clusters were systematically evaluated in the chicken genome and diverse expression patterns for closely linked miRNAs were observed. Our results significantly expand the set of known miRNAs in the chicken and provide the basis for understanding the structural and functional evolution of miRNA genes in vertebrates.