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A novel expression system for artificial miRNAs containing no endogenous miRNA precursor sequences.

ABSTRACT: Recently, artificial microRNA (miRNA)-mediated RNA interference (RNAi) systems have been developed as useful tools to study gene functions. We report an artificial miRNA precursor motif (AMPM) containing several restriction sites in the loop and in the extended stem structures, and generated a vector-based expression system for AMPM under the control of an RNA polymerase II promoter. The AMPM located in the intron or the exon of the selection marker gene mediated silencing of the reporter luciferase gene expression in HeLa cells. Northern blotting and colony formation assays revealed that the AMPM was efficiently and appropriately processed into mature miRNA. The AMPA system also down-regulated endogenous p53 and laminA/C proteins in stable transfectants. Moreover, clustered AMPMs separated by an appropriate spacer, targeting different sites within a single mRNA mediated gene silencing, even if the sequences of the target and the AMPM were partially complementary. This indicates the potential therapeutic utility of clustered AMPMs for highly-mutable targets, such as human immunodeficiency virus type 1.

SUBMITTER: Shibata A

PROVIDER: S-EPMC2737215 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

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A novel expression system for artificial miRNAs containing no endogenous miRNA precursor sequences.

Shibata Atsushi A Iwaki Akiko A Fukumaki Yasuyuki Y

Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research 20070527 1

Recently, artificial microRNA (miRNA)-mediated RNA interference (RNAi) systems have been developed as useful tools to study gene functions. We report an artificial miRNA precursor motif (AMPM) containing several restriction sites in the loop and in the extended stem structures, and generated a vector-based expression system for AMPM under the control of an RNA polymerase II promoter. The AMPM located in the intron or the exon of the selection marker gene mediated silencing of the reporter lucife ...[more]

PMID: 19771221

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Project description:BackgroundThe plant miRNAs represent an important class of endogenous small RNAs that guide cleavage of an mRNA target or repress its translation to control development and adaptation to stresses. MiRNAs are nuclear-encoded genes transcribed by RNA polymerase II, producing a primary precursor that is subsequently processed by DCL1 an RNase III Dicer-like protein. In rice hundreds of miRNAs have been described or predicted, but little is known on their genes and precursors which are important criteria to distinguish them from siRNAs. Here we develop a combination of experimental approaches to detect novel miRNAs in rice, identify their precursor transcripts and genes and predict or validate their mRNA targets.ResultsWe produced four cDNA libraries from small RNA fractions extracted from distinct rice tissues. By in silico analysis we selected 6 potential novel miRNAs, and confirmed that their expression requires OsDCL1. We predicted their targets and used 5'RACE to validate cleavage for three of them, targeting a PPR, an SPX domain protein and a GT-like transcription factor respectively. In addition, we identified precursor transcripts for the 6 miRNAs expressed in rice, showing that these precursors can be efficiently processed using a transient expression assay in transfected Nicotiana benthamiana leaves. Most interestingly, we describe two precursors producing tandem miRNAs, but in distinct arrays. We focus on one of them encoding osa-miR159a.2, a novel miRNA produced from the same stem-loop structure encoding the conserved osa-miR159a.1. We show that this dual osa-miR159a.2-osa-miR159a.1 structure is conserved in distant rice species and maize. Finally we show that the predicted mRNA target of osa-miR159a.2 encoding a GT-like transcription factor is cleaved in vivo at the expected site.ConclusionThe combination of approaches developed here identified six novel miRNAs expressed in rice which can be clearly distinguished from siRNAs. Importantly, we show that two miRNAs can be produced from a single precursor, either from tandem stem-loops or tandemly arrayed in a single stem-loop. This suggests that processing of these precursors could be an important regulatory step to produce one or more functional miRNAs in plants and perhaps coordinate cleavage of distinct targets in the same plant tissue.

Project description:BackgroundmicroRNAs (miRNAs) are implicated in regulation of many cellular processes. miRNAs are processed to their mature functional form in a step-wise manner by multiple proteins and cofactors in the nucleus and cytoplasm. Many miRNAs are conserved across vertebrates. Mature miRNAs have recently been characterized in Atlantic halibut (Hippoglossus hippoglossus L.). The aim of this study was to identify and characterize precursor miRNA (pre-miRNAs) and miRNA targets in this non-model flatfish. Discovery of miRNA precursor forms and targets in non-model organisms is difficult because of limited source information available. Therefore, we have developed a methodology to overcome this limitation.MethodsGenomic DNA and small transcriptome of Atlantic halibut were sequenced using Roche 454 pyrosequencing and SOLiD next generation sequencing (NGS), respectively. Identified pre- miRNAs were further validated with reverse-transcription PCR. miRNA targets were identified using miRanda and RNAhybrid target prediction tools using sequences from public databases. Some of miRNA targets were also identified using RACE-PCR. miRNA binding sites were validated with luciferase assay using the RTS34st cell line.ResultsWe obtained more than 1.3 M and 92 M sequence reads from 454 genomic DNA sequencing and SOLiD small RNA sequencing, respectively. We identified 34 known and 9 novel pre-miRNAs. We predicted a number of miRNA target genes involved in various biological pathways. miR-24 binding to kisspeptin 1 receptor-2 (kiss1-r2) was confirmed using luciferase assay.ConclusionThis study demonstrates that identification of conserved and novel pre-miRNAs in a non-model vertebrate lacking substantial genomic resources can be performed by combining different next generation sequencing technologies. Our results indicate a wide conservation of miRNA precursors and involvement of miRNA in multiple regulatory pathways, and provide resources for further research on miRNA in non-model animals.

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A novel expression system for artificial miRNAs containing no endogenous miRNA precursor sequences.

Publications

A novel expression system for artificial miRNAs containing no endogenous miRNA precursor sequences.

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