Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells

A 21-base pair RNA duplex that perfectly matches an endogenous target mRNA selectively degrades the mRNA and suppresses gene expression in mammalian tissue culture cells. A single base mismatch with the target is believed to protect the mRNA from degradation, making this type of interference highly specific to the targeted gene. A short RNA with mismatches to a target sequence present in multiple copies in the 3'-untranslated region of an exogenously expressed gene can, however, silence it by translational repression. Here we report that a mismatched RNA, targeted to a single site in the coding sequence of an endogenous gene, can efficiently silence gene expression by repressing translation. The antisense strand of such a mismatched RNA requires a 5'-phosphate but not a 3'-hydroxyl group. G.U wobble base pairing is tolerated as a match for both RNA degradation and translation repression. Together, these findings suggest that a small inhibitory RNA duplex can suppress expression of off-target cellular proteins by RNA degradation or translation repression. Proper design of experimental small inhibitory RNAs or a search for targets of endogenous micro-RNAs must therefore take into account that these short RNAs can affect expression of cellular genes with as many as 3-4 base mismatches and additional G.U mismatches.     

Engineering novel traits in plants through RNA interference

RNA interference (RNAi) is a homology-dependent gene silencing technology that involves double-stranded RNA directed against a target gene or its promoter region. Using hairpin constructs, double-stranded RNA can be expressed in plants relatively easily, enabling this technology to be applied to a wide range of species to silence the expression of both specific endogenous genes and genes of invading pathogens. RNAi has also been used to engineer metabolic pathways to overproduce secondary products with health, yield or environmental benefits. The application of tissue-specific or inducible gene silencing, with the use of appropriate promoters, and the ability to silence several genes simultaneously should enhance our ability to create novel traits in plants.     

RNA interference: the story of gene silencing in plants and humans

RNA interference is an exciting field of functional genomics that can silence viral genes. This property of interfering RNA can be used to combat viral diseases of plants as well as animals and humans. It is a short sequence of nucleic acid that can bind to the mRNA of the gene and interferes the process of its expression. It is diverse in occurrence as well as in applications. It occurs from nematodes to fungi and can cause gene silencing in plants, animals and human beings. Small interfering RNAs are used to silence plant viral genes and in production of therapeutic drugs against Hepatitis or Immuno-deficiency viruses in human. In this review, we will discuss the history, mechanism and applications of RNA interference in plant, animal and human research.     

DNA methylation: the nuts and bolts of repression

DNA methylation is an epigenetic modification which plays an important role in chromatin organization and gene expression. DNA methylation can silence genes and repetitive elements through a process which leads to the alteration of chromatin structure. The mechanisms which target DNA methylation to specific sites in the genome are not fully understood. In this review, we will discuss the mechanisms which lead to the long-term silencing of genes and will survey the progression that has been made in determining the targeted mechanisms for de novo DNA methylation.     

Use of bicistronic vectors in combination with flow cytometry to screen for effective small interfering RNA target sequences

The efficacy and specificity of small interfering RNAs (siRNAs) are largely dependent on the siRNA sequence. Since only empirical strategies are currently available for predicting these parameters, simple and accurate methods for evaluating siRNAs are needed. To simplify such experiments, target genes are often tagged with reporters for easier readout. Here, we used a bicistronic vector expressing a target gene and green fluorescent protein (GFP) to create a system in which the effect of an siRNA sequence was reflected in the GFP expression level. Cells were transduced with the bicistronic vector, expression vectors for siRNA and red fluorescent protein (RFP). Flow cytometric analysis of the transduced cells revealed that siRNAs for the target gene silenced GFP from the bicistronic vector, but did not silence GFP transcribed without the target gene sequence. In addition, the mean fluorescence intensities of GFP on RFP-expressing cells correlated well with the target gene mRNA and protein levels. These results suggest that this flow cytometry-based method enables us to quantitatively evaluate the efficacy and specificity of siRNAs. Because of its simplicity and effectiveness, this method will facilitate the screening of effective siRNA target sequences, even in high-throughput applications.     

Validation of RNAi silencing specificity using synthetic genes: salicylic acid-binding protein 2 is required for innate immunity in plants

RNA interference (RNAi) is widely used to specifically silence the expression of any gene to study its function and to identify and validate therapeutic targets. Despite the popularity of this technology, recent studies have shown that RNAi may also silence non-targeted genes. Here we demonstrate the utility of a quick, efficient and robust approach to directly validate the specificity of RNAi as an alternative to indirect validation of RNAi through gene expression profiling. Our approach involves reversing (complementing) the RNAi-induced phenotype by introducing a synthetic version of the target gene that is designed to escape silencing. This synthetic gene complementation approach can also be used for mutational analysis of the target gene, or to provide a functional version of a defective protein after silencing the defective gene by RNAi. Using this approach we demonstrate that the loss of systemic acquired resistance, a form of innate immunity in plants, is indeed due to the silencing of salicylic acid-binding protein 2 rather than to off-target effects.     

Predicting siRNA activity based on back-propagation neural network

RNA interference (RNAi) is a phenomenon of gene silence induced by a double-stranded RNA (dsRNA) homologous to a target gene.RNAi can be used to identify the function of genes or to knock down the targeted genes.In RNAi technology,19 bp double-stranded short interfering RNAs (siRNA) with characteristic 3' overhangs are usually used.The effects of siRNAs are quite varied due to the different choices in the sites of target mRNA.Moreover,there are many factors influencing siRNA activity and these factors are usually nonlinear.To find the motif features and the effect on siRNA activity,we carried out a feature extraction on some published experimental data and used these features to train a backpropagation neural network (BP NN).Then,we used the trained BP NN to predict siRNA activity.     

Identification of Homeotic Target Genes in Drosophila Melanogaster Including Nervy, a Proto-Oncogene Homologue

In Drosophila, the specific morphological characteristics of each segment are determined by the homeotic genes that regulate the expression of downstream target genes. We used a subtractive hybridization procedure to isolate activated target genes of the homeotic gene Ultrabithorax (Ubx). In addition, we constructed a set of mutant genotypes that measures the regulatory contribution of individual homeotic genes to a complex target gene expression pattern. Using these mutants, we demonstrate that homeotic genes can regulate target gene expression at the start of gastrulation, suggesting a previously unknown role for the homeotic genes at this early stage. We also show that, in abdominal segments, the levels of expression for two target genes increase in response to high levels of Ubx, demonstrating that the normal down-regulation of Ubx in these segments is functional. Finally, the DNA sequence of cDNAs for one of these genes predicts a protein that is similar to a human proto-oncogene involved in acute myeloid leukemias. These results illustrate potentially general rules about the homeotic control of target gene expression and suggest that subtractive hybridization can be used to isolate interesting homeotic target genes.     

Predicting siRNA activity based on back-propagation neural network

RNA interference (RNAi) is a phenomenon of gene silence induced by a double-stranded RNA (dsRNA) homologous to a target gene. RNAi can be used to identify the function of genes or to knock down the targeted genes. In RNAi technology, 19 bp double-stranded short interfering RNAs (siRNA) with characteristic 39 overhangs are usually used. The effects of siRNAs are quite varied due to the different choices in the sites of target mRNA. Moreover, there are many factors influencing siRNA activity and these factors are usually nonlinear. To find the motif features and the effect on siRNA activity, we carried out a feature extraction on some published experimental data and used these features to train a back-propagation neural network (BP NN). Then, we used the trained BP NN to predict siRNA activity. __________ Translated from Acta Biophysica Sinica, 2006, 22(6): 429–434 [译自: 生物物理学报]     

Artificial microRNAs (amiRNAs) engineering - On how microRNA-based silencing methods have affected current plant silencing research

In recent years, endogenous microRNAs have been described as important regulators of gene expression in eukaryotes. Artificial microRNAs (amiRNAs) represent a recently developed miRNA-based strategy to silence endogenous genes. amiRNAs can be created by exchanging the miRNA/miRNA^∗ sequence within a miRNA precursor with a sequence designed to match the target gene, this is possible as long as the secondary RNA structure of the precursor is kept intact. In this review, we summarize the basic methodologies to design amiRNAs and detail their applications in plants genetic functional studies as well as their potential for crops genetic improvement.     

Enzymatic production and expression of shRNAmir30 from cDNAs

RNA interference (RNAi) is an important tool for studying gene function and genetic networks. Double-stranded RNA (dsRNA) triggers RNAi that selectively silences gene expression mainly by degrading target mRNA sequences. Short interfering RNA, short hairpin RNA (shRNA), long dsRNA, and microRNA-based shRNA (shRNAmir) are four different types of dsRNA that have been widely used to silence gene expression in cultured cells, tissues, organs, and organisms. Long dsRNAs are usually 200–500 nucleotides in length and can selectively suppress expression of target genes in Caenorhabditis elegans and Drosophila but not in mammals due to unwanted non-specific knockdown. Thus, multiple attempts have been made to synthesize, express, and deliver short dsRNAs that specifically silence target genes in mammals. We describe a method for constructing an RNAi library by converting cDNAs into shRNAmir30 sequences by sequential treatment with different enzymes and affinity purification of biotin- or digoxygenin-labeled DNA fragments. We also developed a system to generate stable cell lines that uniformly express shRNAmir30s and fluorescence reporters by Cre recombinase-dependent site-specific recombination. Thus, combined with the RNAi library, this system facilitates screening for potent RNAi sequences that strongly suppress expression of target genes.     

Silencing of Six Hydrophobins in Cladosporium fulvum: Complexities of Simultaneously Targeting Multiple Genes

In this study, we have constructed and expressed inverted repeat chimeras from the first exons of the six known hydrophobins of the fungus Cladosporium fulvum, the causal agent of tomato leaf mold. We used quantitative PCR to measure specifically the expression levels of the hydrophobins. The targeted genes are silenced to different degrees, but we also detected clear changes in the expression levels of nontargeted genes. This work highlights the difficulties that are likely to be encountered when attempting to silence more than one gene in a multigene family.     

A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing

Virus-induced gene silencing (VIGS) is a rapid and robust method for determining and studying the function of plant genes or expressed sequence tags (ESTs). However, only a few plant species are amenable to VIGS. There is a need for a systematic study to identify VIGS-efficient plant species and to determine the extent of homology required between the heterologous genes and their endogenous orthologs for silencing. Two approaches were used. First, the extent of phytoene desaturase (PDS) gene silencing was studied in various Solanaceous plant species using Nicotiana benthamiana NbPDS sequences. In the second approach, PDS sequences from a wide range of plant species were used to silence the PDS gene in N. benthamiana. The results showed that tobacco rattle virus (TRV)-mediated VIGS can be performed in a wide range of Solanaceous plant species and that heterologous gene sequences from far-related plant species can be used to silence their respective orthologs in the VIGS-efficient plant N. benthamiana. A correlation was not always found between gene silencing efficiency and percentage homology of the heterologous gene sequence with the endogenous gene sequence. It was concluded that a 21-nucleotide stretch of 100% identity between the heterologous and endogenous gene sequences is not absolutely required for gene silencing.     

Human immunodeficiency virus type 1 escape is restricted when conserved genome sequences are targeted by RNA interference

RNA interference (RNAi) is a cellular mechanism in which small interfering RNAs (siRNAs) mediate sequence-specific gene silencing by cleaving the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short-hairpin RNAs (shRNAs). We previously reported efficient HIV-1 inhibition by an shRNA against the nonessential nef gene but also described viral escape by mutation or deletion of the nef target sequence. The objective of this study was to obtain insight in the viral escape routes when essential and highly conserved sequences are targeted in the Gag, protease, integrase, and Tat-Rev regions of HIV-1. Target sequences were analyzed of more than 500 escape viruses that were selected in T cells expressing individual shRNAs. Viruses acquired single point mutations, occasionally secondary mutations, but—in contrast to what is observed with nef—no deletions were detected. Mutations occurred predominantly at target positions 6, 8, 9, 14, and 15, whereas none were selected at positions 1, 2, 5, 18, and 19. We also analyzed the type of mismatch in the siRNA-target RNA duplex, and G-U base pairs were frequently selected. These results provide insight into the sequence requirements for optimal RNAi inhibition. This knowledge on RNAi escape may guide the design and selection of shRNAs for the development of an effective RNAi therapy for HIV-1 infections.     

基于BP神经网络的siRNA活性预测

RNA干扰(RNAinterference,RNAi)是由双链RNA(dsRNA)引起的基因沉默现象,它通过降解具有同源序列的mRNA来起作用,特殊设计的siRNA能使靶基因发生特异性沉默,起到确定基因功能或沉默致病基因从而治疗疾病的目的。在RNAi技术的应用中,通常采用的是长度为19bp,正、反义链3'端各有2个不配对碱基的双链RNA(siRNA)。但针对靶基因不同位点设计的siRNA作用效果差别很大。影响siRNA效果的因素是多方面的,这些因素的作用又是非线性的。本文在研究影响siRNA作用效果的各种因素的基础上,对已经公开发表的实验数据进行特征提取,作为BP神经网络的训练数据,并将训练好的BP神经网络用于siRNA活性预测。