PTPome-wide functional RNA interference screening methods

The elucidation of the entire complement of genes encoding protein tyrosine phosphatases (PTPs) in human genome, the human 'PTPome', has made it possible to experimentally address the entire family in an unbiased manner. Here we describe a functional RNA interference-based assay, in which we evaluate 87 of the known 107 PTPs for effects on cell survival in a high throughput manner. The details of assay rationale and design, instrumentation, pitfalls, data analysis, and further validation steps are described. We also discuss the suitability of this technology for further assay development and application to other functional read-outs and signaling pathways.     

Structural and functional insights into human Tudor-SN, a key component linking RNA interference and editing

Human Tudor-SN is involved in the degradation of hyper-edited inosine-containing microRNA precursors, thus linking the pathways of RNA interference and editing. Tudor-SN contains four tandem repeats of staphylococcal nuclease-like domains (SN1–SN4) followed by a tudor and C-terminal SN domain (SN5). Here, we showed that Tudor-SN requires tandem repeats of SN domains for its RNA binding and cleavage activity. The crystal structure of a 64-kD truncated form of human Tudor-SN further shows that the four domains, SN3, SN4, tudor and SN5, assemble into a crescent-shaped structure. A concave basic surface formed jointly by SN3 and SN4 domains is likely involved in RNA binding, where citrate ions are bound at the putative RNase active sites. Additional modeling studies provide a structural basis for Tudor-SN's preference in cleaving RNA containing multiple I·U wobble-paired sequences. Collectively, these results suggest that tandem repeats of SN domains in Tudor-SN function as a clamp to capture RNA substrates.     

RNA干涉的研究进展

生物体内导入双链RNA后会引起体内同源基因特异性的沉默,这种现象称为RNA干涉,本主要介绍RNA干涉的研究历史,作用机制和应用等方面的情况。     

RNA干涉技术

RNA干涉(RNAi)技术是利用一些小的双链RNA来高效、特异地阻断体内特定基因的表达,并促使mRNA降解,从而诱使细胞表现出特定基因缺失的表型。本从RNAi技术的历史、作用机制、研究策略、研究现状及应用前景等几个方面进行了综述,预测RNAi将会给基因治疗的发展带来新的希望。     

RNA干扰的研究进展

RNA干扰是指外源双链RNA进入细胞后引起与其同源的mRNA特异性降解的现象,它是真核生物在长期进化中形成的一种保守的防御机制,对真核生物有着重要的意义,它参与真核生物抵御病毒侵染、阻断转座子的异常活动,调控基因表达。RNA干扰已成为一种进行基因功能分析的强有力的工具,并有望成为最有潜力的基因干预治疗方法。     

RNA interference in cancer

In the recent years, RNA interference (RNAi) has emerged as a major regulatory mechanism in eukaryotic gene expression. The realization that changes in the levels of microRNAs are directly associated with cancer led to the recognition of a new class of tumor suppressors and oncogenes. Moreover, RNAi has been turned into a potent tool for artificially modulating gene expression through the introduction of short interfering RNAs. A plethora of individual inhibitory RNAs as well as several large collections of these reagents have been generated. The systems for stable and regulated expression of these molecules emerged as well. These tools have helped to delineate the roles of various cellular factors in oncogenesis and tumor suppression and laid the foundation for new approaches in gene discovery. Furthermore, successful inhibition of tumor cell growth by RNAi aimed at oncogenes in vitro and in vivo supports the enthusiasm for potential therapeutic applications of this technique. In this article we review the evidence of microRNA involvement in cancer, the use of short interfering RNAs in forward and reverse genetics of this disease, and as well as both the benefits and limitations of experimental RNAi.     

The functional consequence of RhoA knockdown by RNA interference in rat cerebral arteries

Uridine triphosphate (UTP) constricts cerebral arteries by activating transduction pathways that increase cytosolic [Ca(2+)] and myofilament Ca(2+) sensitivity. The signaling proteins that comprise these pathways remain uncertain with recent studies implicating a role for several G proteins. To start clarifying which G proteins enable UTP-induced vasoconstriction, a small interfering RNA (siRNA) approach was developed to knock down specified targets in rat cerebral arteries. siRNA directed against G(q) and RhoA was introduced into isolated cerebral arteries using reverse permeabilization. Following a defined period of organ culture, arteries were assayed for contractile function, mRNA levels, and protein expression. Targeted siRNA reduced RhoA or G(q) mRNA expression by 60-70%, which correlated with a reduction in RhoA but not G(q) protein expression. UTP-induced constriction was abolished in RhoA-depleted arteries, but this was not due to a reduction in myosin light chain phosphorylation. UTP-induced actin polymerization was attenuated in RhoA-depleted arteries, which would explain the loss of agonist-induced constriction. In summary, this study illustrates that siRNA approaches can be effectively used on intact arteries to induce targeted knockdown given that the protein turnover rate is sufficiently high. It also demonstrates that the principal role of RhoA in agonist-induced constriction is to facilitate the formation of F-actin, the physical structure to which phosphorylated myosin binds to elicit arterial constriction.     

Recent approaches to probe functional groups in ribonuclease P RNA by modification interference

Modification interference is a powerful method to identify important functional groups in RNA molecules. We review here recent developments of techniques to screen for chemical modifications that interfere with (i) binding of(pre-)tRNA to bacterial RNase P RNA or (ii) pre-tRNA cleavage by this ribozyme. For example, two studies have analyzed positions at which a substitution of sulfur for thepro-Rp oxygen affects tRNA binding [1] or catalysis [2]. The results emphasize the functional key role of a central core element present in all known RNase P RNA subunits. The four sulfur substitutions identified in one study [2] to inhibit the catalytic step also interfered with binding of tRNA toE. coli RNase P RNA [1]. This suggests that losses in binding energy due to the modification at these positions affect the enzyme-substrate and the enzyme-transition state complex. In addition, the two studies have revealed, for the first time, sites of direct metal ion coordination in RNase P RNA. The potentials, limitations and interpretational ambiguities of modification interference experiments as well as factors influencing their outcome are discussed.Abbreviations nt nucleotide(s) - PAGE polyacrylamide gel electrophoresis     

Environmental RNA interference

The discovery of RNA interference (RNAi), the process of sequence-specific gene silencing initiated by double-stranded RNA (dsRNA), has broadened our understanding of gene regulation and has revolutionized methods for genetic analysis. A remarkable property of RNAi in the nematode Caenorhabditis elegans and in some other multicellular organisms is its systemic nature: silencing signals can cross cellular boundaries and spread between cells and tissues. Furthermore, C. elegans and some other organisms can also perform environmental RNAi: sequence-specific gene silencing in response to environmentally encountered dsRNA. This phenomenon has facilitated significant technological advances in diverse fields including functional genomics and agricultural pest control. Here, we describe the characterization and current understanding of environmental RNAi and discuss its potential applications.     

RNA干扰与技术

RNA干扰(RNA interference,RNAi)是由双链RNA诱导的、序列特异的基因沉默机制。它是自然存在于植物、线虫和果蝇中抵抗外来基因(包括病毒、转座子)入侵的方式。在哺乳动物细胞中,能够人工诱导RNA干扰,沉默有同源序列基因表达。这一新技术具有特异性、高效性。因此,正被用来研究人类基因组的功能、肿瘤和抗病毒感染等。