MicroRNAs and the Operon paper

The “Operon paper” by F. Jacob and J. Monod started 50 years of research into understanding how the expression of genes is regulated on a molecular level. Ten years ago, microRNAs (miRNAs) emerged as major regulators of eukaryotic gene expression. Here, I will review the basic principles of gene regulation by miRNAs and how these principles can be linked to insights from the Operon paper. A lot of what is understood about miRNAs required a combination of computational and experimental methods. I will discuss some examples that illustrate the power of this approach.     

Deep sequencing analysis of small non-coding RNAs reveals the diversity of microRNAs and piRNAs in the human epididymis

The epididymis plays a crucial role in regulating the development of sperm motility and fertilizing capacity. Small non-coding RNAs (sncRNAs), especially microRNAs (miRNAs), can participate in the regulation of various physiological pathways. However, their abundance and whether they are involved in the regulation of gene expression in the human epididymis are unknown. By adopting the Solexa deep sequencing approach, we systematically investigated the sncRNAs in the adult human epididymis. A total of 4903 unique sequences representing 527 known miRNA were discovered. Eighteen novel miRNA genes encoding 23 mature miRNAs were also identified and the expression of some of them was confirmed by qRT-PCR. The presence of Piwi-interacting RNAs (piRNAs) in the library also adds to the diversity of the sncRNA population in the human epididymis. This research will contribute to a preliminary database for their functional study in male reproductive system.     

RNA interference induced by siRNAs modified with 4'-thioribonucleosides in cultured mammalian cells

Short interfering RNAs (siRNAs) variously modified with 4'-thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense-strand were well tolerated for RNAi activity except for full modification with 4'-thioribonucleosides. However, the activity of siRNAs modified at the antisense-strand was dependent on the position and the number of modifications with 4'-thioribonucleosides. Since modifications of siRNAs with 4'-thioribonucleosides were well tolerated in RNAi activity compared with that of 2'-O-methyl nucleosides, 4'-thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs.     

MicroRNAs in Amoebozoa: Deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs

The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a shared origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. Micro (mi)RNAs regulate protein-coding genes and play vital roles in plants and animals, but less is known about their functions in other organisms. Here, we report, for the first time, deep sequencing of small RNAs from the social amoeba Dictyostelium discoideum. RNA from growing single-cell amoebae as well as from two multicellular developmental stages was sequenced. Computational analyses combined with experimental data reveal the expression of miRNAs, several of them exhibiting distinct expression patterns during development. To our knowledge, this is the first report of miRNAs in the Amoebozoa supergroup. We also show that overexpressed miRNA precursors generate miRNAs and, in most cases, miRNA* sequences, whose biogenesis is dependent on the Dicer-like protein DrnB, further supporting the presence of miRNAs in D. discoideum. In addition, we find miRNAs processed from hairpin structures originating from an intron as well as from a class of repetitive elements. We believe that these repetitive elements are sources for newly evolved miRNAs.     

RNA干扰作用(RNAi)研究进展

RNA干扰作用 (RNAi)是生物界一种古老而且进化上高度保守的现象 ,是基因转录后沉默作用 (PTGS)的重要机制之一 .RNAi主要通过dsRNA被核酸酶切割成 2 1～ 2 5nt的干扰性小RNA即siRNA ,由siRNA介导识别并靶向切割同源性靶mRNA分子而实现 .RNAi要有多种蛋白因子以及ATP参与 ,而且具有生物催化反应特征 .RNAi是新发现的一种通过dsRNA介导的特异性高效抑制基因表达途径 ,在后基因组时代的基因功能研究和药物开发中具有广阔应用前景     

Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I

Many high-throughput small RNA next-generation sequencing protocols use 5′ preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter''s 5′ end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5′ adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3′ blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3′ ends to be 5′ adenylylated at >90% efficiency.     

Dicing and slicing: the core machinery of the RNA interference pathway

RNA interference (RNAi) is broadly defined as a gene silencing pathway that is triggered by double-stranded RNA (dsRNA). Many variations have been described on this theme. The dsRNA trigger can be supplied exogenously, as an experimental tool, or can derive from the genome in the form of microRNAs. Gene silencing can be the result of nucleolytic degradation of the mRNA, or by translational suppression. At the heart of the pathway are two ribonuclease machines. The ribonuclease III enzyme Dicer initiates the RNAi pathway by generating the active short interfering RNA trigger. Silencing is effected by the RNA-induced silencing complex and its RNaseH core enzyme Argonaute. This review describes the discovery of these machines and discusses future lines of work on this amazing biochemical pathway.     

核仁小RNA源性小RNA

最新研究结果表明,一些与RNA介导基因沉默相关的小RNA由核仁小RNA(small nucleolar RNA,snoRNA)加工产生,这种小RNA被称为核仁小RNA源性小RNA(snoRNA derived small RNA,sdRNA)。sdRNA现象分布物种广;涉及的snoRNA种类全,数量多;产生的小RNA分子大小不一、数量、种类多。表明这种小RNA在生物中存在着广泛的普遍性。sdRNA的发现拓展了snoRNA的功能,揭示了snoRNA与RNA介导的基因沉默之间的紧密关系,增强了snoRNA在RNA调控网络中的重要性,并为进一步研究RNA调控网络开启了一扇门。     

RNA helicase A is not required for RISC activity

It has been shown that siRNAs can compete with each other or with endogenous miRNAs for RISC components. This competition may complicate the interpretations of phenotypes observed through siRNA-mediated knockdown of genes, especially those genes implicated in the RISC pathway. In this study, we re-examined the function of RNA helicase A (RHA), which has been previously proposed to function in RISC loading based on siRNA-mediated knockdown studies. Here we show that reduced RISC activity or loading of siRNAs was observed only in cells depleted of RHA using siRNA, but not using RNaseH-dependent antisense oligonucleotides (ASOs), suggesting that the impaired RISC function stems from the competition between pre-existing and newly transfected siRNAs, but not from reduction of the RHA protein. This view is further supported by the findings that cells depleted of a control protein, NCL1, using siRNA, but not ASO, exhibited similar defects on the loading and activity of a subsequently transfected siRNA. Transfection of RHA or NCL1 siRNAs, but not ASOs, reduced the levels of endogenous miRNAs, suggesting a competition mechanism. As a positive control, we showed that reduction of MOV10 by either siRNA or ASO decreased siRNA activity, confirming its role in RISC function. Together, our results indicate that RHA is not required for RISC activity or loading, and suggest that proper controls are required when using siRNAs to functionalize genes to avoid competition effects.     

Substrate-specific kinetics of Dicer-catalyzed RNA processing

The specialized ribonuclease Dicer plays a central role in eukaryotic gene expression by producing small regulatory RNAs—microRNAs (miRNAs) and short interfering RNAs (siRNAs)—from larger double-stranded RNA (dsRNA) substrates. Although Dicer will cleave both imperfectly base-paired hairpin structures (pre-miRNAs) and perfect duplexes (pre-siRNAs) in vitro, it has not been clear whether these are mechanistically equivalent substrates and how dsRNA binding proteins such as trans-activation response (TAR) RNA binding protein (TRBP) influence substrate selection and RNA processing efficiency. We show here that human Dicer is much faster at processing a pre-miRNA substrate compared to a pre-siRNA substrate under both single and multiple turnover conditions. Maximal cleavage rates (V_max) calculated by Michaelis-Menten analysis differed by more than 100-fold under multiple turnover conditions. TRBP was found to enhance dicing of both substrates to similar extents, and this stimulation required the two N-terminal dsRNA binding domains of TRBP. These results demonstrate that multiple factors influence dicing kinetics. While TRBP stimulates dicing by enhancing the stability of Dicer-substrate complexes, Dicer itself generates product RNAs at rates determined at least in part by the structural properties of the substrate.     

RNA干扰机制研究进展

RNAi是多种生物体内由dsRNA介导的同源mRNA降解现象。这是一个高度特异化的过程,涉及多种蛋白质的共同参与。在这一过程中,siRNA的结构影响其两条链装配到RISC中去的能力。除了与RISC结合外,siRNA还引导了RITS复合物结合到同源染色质,介导异染色质化过程。干扰效应的扩散,即系统性沉默可能依赖于跨膜蛋白的转运,并且很可能是在多因素调控下完成的。Abstract: RNA interference (RNAi) is a phenomenon that the double-stranded RNA (dsRNA) intermediates the degradation of complementary mRNA found in many organisms. This is a specifically mechanism involved in kinds of proteins to complete the interference function. Structure of siRNA affects which strand will be assembled into RISC. Another role of siRNA is directing RITS complex to bind with homologue chromosome, and then induces heterochromatinization. Although systemic silence induced by dsRNA is observed in Caenorhabditis elegans and plants, this progress is probably transmembrane protein-dependent, and mostly, the systemic silencing is controlled by multi-factors.