Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii

MicroRNAs (miRNAs) are small RNAs, 21 to 22 nucleotides long, with important regulatory roles. They are processed from longer RNA molecules with imperfectly matched foldback regions and they function in modulating the stability and translation of mRNA. Recently, we and others have demonstrated that the unicellular alga Chlamydomonas reinhardtii , like diverse multicellular organisms, contains miRNAs. These RNAs resemble the miRNAs of land plants in that they direct site-specific cleavage of target mRNA with miRNA-complementary motifs and, presumably, act as regulatory molecules in growth and development. Utilizing these findings we have developed a novel artificial miRNA system based on ligation of DNA oligonucleotides that can be used for specific high-throughput gene silencing in green algae.     

Technique review: how to use RNA interference.

RNA interference (RNAi) has been rapidly adopted as a general method for inhibiting gene expression in most laboratory organisms. This paper discusses how libraries of RNAi reagents are being used to perform genome-wide reverse genetic screens in both model organisms and mammalian cells. Guidelines for designing effective small interfering RNAs and appropriate controls for mammalian RNAi experiments will also be discussed.     

Gene Discovery and Functional Analyses in the Model Plant Arabidopsis

The present mini-review describes newer methods and strategies, including transposon and T-DNA insertions, TILLING, Deleteagene, and RNA interference, to functionally analyze genes of interest in the model plant Arabidopsis. The relative advantages and disadvantages of the systems are also discussed.     

RNA沉默技术及其在植物中的应用

RNA沉默是广泛存在于生物中的一种古老现象, 是生物抵抗异常DNA的一种保护机制, 同时在生物生长发育过程中扮演着基因表达调控的角色。文章对RNA沉默研究中的几个热点问题进行了介绍, 按RNA沉默持续时间的不同对其在植物中应用的方法进行了分析, 并重点论述了RNA沉默在植物功能基因组研究、作物品质改良以及抗病毒植物培育等方面的应用及其发展前景。     

MIGS: miRNA-induced gene silencing

Gene silencing is an important tool in the study of gene function. Virus-induced gene silencing (VIGS) and hairpin RNA interference (hpRNAi), both of which rely on small interfering RNAs, together with artificial microRNAs (amiRNA), are amongst the most popular methods for reduction of gene activity in plants. However, all three approaches have limitations. Here, we introduce miRNA-induced gene silencing (MIGS). This method exploits a special 22-nucleotide miRNA of Arabidopsis thaliana, miR173, which can trigger production of another class of small RNAs called trans-acting small interfering RNAs (tasiRNAs). We show that fusion of gene fragments to an upstream miR173 target site is sufficient for effective silencing of the corresponding endogenous gene. MIGS can be reliably used for the knockdown of a single gene or of multiple unrelated genes. In addition, we show that MIGS can be applied to other species by co-expression of miR173.     

Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway

In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character—as is found in small interfering RNAs (siRNAs)—directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient loading of miRNA* strands in Ago2 favors duplexes with a paired central region and requires both Dcr-2 and R2D2. Those miRNA and miRNA* sequences bound to Ago2, like siRNAs diced in vivo from long double-stranded RNA, typically begin with cytidine, whereas Ago1-bound miRNA and miRNA* disproportionately begin with uridine. Consequently, some pre-miRNA generate two or more isoforms from the same side of the stem that differentially partition between Ago1 and Ago2. Our findings provide the first genome-wide test for the idea that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide.     

Identification and characterization of small RNAs involved in RNA silencing

Double-stranded RNA (dsRNA) is a potent trigger of sequence-specific gene silencing mechanisms known as RNA silencing or RNA interference. The recognition of the target sequences is mediated by ribonucleoprotein complexes that contain 21- to 28-nucleotide (nt) guide RNAs derived from processing of the trigger dsRNA. Here, we review the experimental and bioinformatic approaches that were used to identify and characterize these small RNAs isolated from cells and tissues. The identification and characterization of small RNAs and their expression patterns is important for elucidating gene regulatory networks.     

病毒microRNA研究进展

microRNA(miRNA)是一类存在于多细胞生物中长约21-24nt的非编码RNA分子,它们与靶mRNA分子互补结合抑制蛋白翻译或导致mRNA降解,从而调控靶基因表达。miRNA已被证实在多种代谢途径中发挥重要作用,调节包括细胞分化和分裂、细胞凋亡及癌症发生在内的多个细胞过程。利用生物信息学以及分子克隆的方法在线虫、哺乳动物以及植物中已发现超过4000条miRNA。最近在病毒中也发现有miRNA基因存在,通过对病毒miRNA靶基因的预测,推测其在病毒复制过程中发挥重要的调控作用。目前病毒编码的miRNA分子的特点、转录机制、功能、进化保守性以及病毒与宿主miRNA的关系都已有一定的了解。对于病毒相关miRNA研究的深入,必将对认识病毒-宿主相互作用以及相关疾病的治疗带来新的启示。     

Effective suppression of HIV-1 by artificial bispecific miRNA targeting conserved sequences with tolerance for wobble base-pairing

The high genetic diversity and mutability of HIV pose a major problem for RNAi-mediated antiviral therapy. Simultaneous targeting of multiple highly conserved viral sequences has been suggested for durable cross-clade inhibition. Here we validate the approach of co-targeting two conserved sequences in the Tat and Vif genes. When coexpressed as artificial microRNA from a PolII driven miR-155-based vector, the sequences together mediated effective and sustained inhibition of HIV replication without virus breakout. To understand the nature of this efficient control, we analyzed genome sequences of 625 HIV-1 isolates in the Los Alamos Sequence database. Interestingly most natural variants were capable of wobble binding with the Tat/Vif siRNAs. Efficient silencing of reporter luciferase constructs bearing these variants residues verified that the Tat/Vif sequences together tolerated wobble binding and mediated functional RNAi. We propose the rationale of targeting highly conserved HIV sequences where wobble substitutions permit functional RNAi for global HIV repression.     

Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs

Hybrid organisms may fail to develop, be sterile or they may be more vigorous than either of the parents. Examples of hybrid vigour or hybrid necrosis in the F1 are often not inherited stably in subsequent generations if they are associated with overdominance. There can also be transgressive phenotypes that are inherited stably in these later generations, but the underlying mechanisms are not well understood. Here we have investigated the possibility that stable transgressive phenotypes in the progeny of crosses between cultivated tomato (Solanum lycopersicum cv. M82) and a wild relative (Solanum pennellii, accession LA716) are associated with micro or small interfering(si) RNAs. We identified loci from which these small(s)RNAs were more abundant in hybrids than in either parent and we show that accumulation of such transgressive sRNAs correlated with suppression of the corresponding target genes. In one instance this effect was associated with hypermethylation of the corresponding genomic DNA. Our results illustrate a potential role of transgressive sRNAs in plant breeding and in natural evolution with wild plants.     

Gene silencing by systemic delivery of synthetic siRNAs in adult mice

In mammalian cells, RNA duplexes of 21-23 nucleotides, known as small interfering RNAs (siRNAs) specifically inhibit gene expression in vitro. Here, we show that systemic delivery of siRNAs can inhibited exogenous and endogenous gene expression in adult mice. Cationic liposome-based intravenous injection in mice of plasmid encoding the green fluorescent protein (GFP) with its cognate siRNA, inhibited GFP gene expression in various organs. Furthermore, intraperitoneal injection of anti-TNF-alpha siRNA inhibited lipopolysaccharide-induced TNF-alpha gene expression, whereas secretion of IL1-alpha was not inhibited. Importantly, the development of sepsis in mice following a lethal dose of lipopolysaccharide injection, was significantly inhibited by pre-treatment of the animals with anti-TNF-alpha siRNAs. Collectively, these results demonstrate that synthetic siRNAs can function in vivo as pharmaceutical drugs.