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Designing small multiple-target artificial RNAs.

ABSTRACT: MicroRNAs (miRNAs) are naturally occurring small RNAs that regulate the expression of several genes. MiRNAs' targeting rules are based on sequence complementarity between their mature products and targeted genes' mRNAs. Based on our present understanding of those rules, we developed an algorithm to design artificial miRNAs to target simultaneously a set of predetermined genes. To validate in silico our algorithm, we tested different sets of genes known to be targeted by a single miRNA. The algorithm finds the seed of the corresponding miRNA among the solutions, which also include the seeds of new artificial miRNA sequences potentially capable of targeting these genes as well. We also validated the functionality of some artificial miRNAs designed to target simultaneously members of the E2F family. These artificial miRNAs reproduced the effects of E2Fs inhibition in both normal human fibroblasts and prostate cancer cells where they inhibited cell proliferation and induced cellular senescence. We conclude that the current miRNA targeting rules based on the seed sequence work to design multiple-target artificial miRNAs. This approach may find applications in both research and therapeutics.

SUBMITTER: De Guire V

PROVIDER: S-EPMC2910070 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Designing small multiple-target artificial RNAs.

De Guire Vincent V Caron Maxime M Scott Nicolas N Ménard Catherine C Gaumont-Leclerc Marie-France MF Chartrand Pascal P Major François F Ferbeyre Gerardo G

Nucleic acids research 20100507 13

MicroRNAs (miRNAs) are naturally occurring small RNAs that regulate the expression of several genes. MiRNAs' targeting rules are based on sequence complementarity between their mature products and targeted genes' mRNAs. Based on our present understanding of those rules, we developed an algorithm to design artificial miRNAs to target simultaneously a set of predetermined genes. To validate in silico our algorithm, we tested different sets of genes known to be targeted by a single miRNA. The algor ...[more]

PMID: 20453028

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Project description:BackgroundRNA silencing is an important mechanism for regulation of endogenous gene expression and defense against genomic intruders in plants. This natural defense system was adopted to generate virus-resistant plants even before the mechanism of RNA silencing was unveiled. With the clarification of that mechanism, transgenic antiviral plants were developed that expressed artificial virus-specific hairpin RNAs (hpRNAs) or microRNAs (amiRNAs) in host plants. Previous works also showed that plant-mediated RNA silencing technology could be a practical method for constructing insect-resistant plants by expressing hpRNAs targeting essential genes of insects.Methodology/principal findingsIn this study, we chose aphid Myzus persicae of order Hemiptera as a target insect. To screen for aphid genes vulnerable to attack by plant-mediated RNA silencing to establish plant aphid resistance, we selected nine genes of M. persicae as silencing targets, and constructed their hpRNA-expressing vectors. For the acetylcholinesterase 2 coding gene (MpAChE2), two amiRNA-expressing vectors were also constructed. The vectors were transformed into tobacco plants (Nicotiana tabacum cv. Xanti). Insect challenge assays showed that most of the transgenic plants gained aphid resistance, among which those expressing hpRNAs targeting V-type proton ATPase subunit E-like (V-ATPaseE) or tubulin folding cofactor D (TBCD) genes displayed stronger aphicidal activity. The transgenic plants expressing amiRNAs targeting two different sites in the MpAChE2 gene exhibited better aphid resistance than the plants expressing MpAChE2-specific hpRNA.Conclusions/significanceOur results indicated that plant-mediated insect-RNA silencing might be an effective way to develop plants resistant to insects with piercing-sucking mouthparts, and both the selection of vulnerable target genes and the biogenetic type of the small RNAs were crucial for the effectiveness of aphid control. The expression of insect-specific amiRNA is a promising and preferable approach to engineer plants resistant to aphids and, possibly, to other plant-infesting insects.

Project description:Many herpesviruses express small noncoding RNAs (sncRNAs), including microRNAs (miRNAs), that may play roles in regulating lytic and latent infections. None have yet been reported in varicella-zoster virus (VZV; also known as human herpesvirus 3 [HHV-3]). Here we analyzed next-generation sequencing (NGS) data for small RNAs in VZV-infected fibroblasts and human embryonic stem cell-derived (hESC) neurons. Two independent bioinformatics analyses identified more than 20 VZV-encoded 20- to 24-nucleotide RNAs, some of which are predicted to have stem-loop precursors potentially representing miRNAs. These sequences are perfectly conserved between viruses from three clades of VZV. One NGS-identified sequence common to both bioinformatics analyses mapped to the repeat regions of the VZV genome, upstream of the predicted promoter of the immediate early gene open reading frame 63 (ORF63). This miRNA candidate was detected in each of 3 independent biological repetitions of NGS of RNA from fibroblasts and neurons productively infected with VZV using TaqMan quantitative PCR (qPCR). Importantly, transfected synthetic RNA oligonucleotides antagonistic to the miRNA candidate significantly enhanced VZV plaque growth rates. The presence of 6 additional small noncoding RNAs was also verified by TaqMan qPCR in productively infected fibroblasts and ARPE19 cells. Our results show VZV, like other human herpesviruses, encodes several sncRNAs and miRNAs, and some may regulate infection of host cells.IMPORTANCE Varicella-zoster virus is an important human pathogen, with herpes zoster being a major health issue in the aging and immunocompromised populations. Small noncoding RNAs (sncRNAs) are recognized as important actors in modulating gene expression, and this study demonstrates the first reported VZV-encoded sncRNAs. Many are clustered to a small genomic region, as seen in other human herpesviruses. At least one VZV sncRNA was expressed in productive infection of neurons and fibroblasts that is likely to reduce viral replication. Since sncRNAs have been suggested to be potential targets for antiviral therapies, identification of these molecules in VZV may provide a new direction for development of treatments for painful herpes zoster.

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Designing small multiple-target artificial RNAs.

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Designing small multiple-target artificial RNAs.

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