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

https://doi.org/10.1016/j.bbrc.2011.02.045Get rights and content

Abstract

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.

Research highlights

► Overview of artificial microRNAs (amiRNAs) designing strategy has been discussed. ► Innovations in model organism has been discussed. ► Novelty in plant immunity and crop improvement has been discussed.

Introduction

Eukaryotes use small non-coding RNAs in processes of post-transcriptional gene silencing (PTGS) to regulate gene expression, as well as to direct epigenetic modifications. These small RNAs interact with silencing complexes to recognize and modify complementary nucleic acids [1], [2].

Small-interfering RNAs (siRNAs, 21–24 nucleotides long) and microRNAs (miRNAs, 21–22 nucleotides long) are the two types of small RNAs involved in PTGS with important differences in their biogenesis and target genes. siRNAs originate commonly from invading or aberrant nucleic acids and produce cis-acting silencing by targeting the same molecule from which they were derived. miRNAs instead originate from endogenous genes and are involved in trans-acting silencing of other endogenous genes [3], [1]. miRNAs are usually transcribed by RNA polymerase II as long primary RNA transcripts (pri-miRNAs), which are normally capped at the 5′ end and polyadenylated at the 3′ end [4]. The pri-miRNA is processed in the nucleus by Dicer-like1 (DCL1) in plants, to liberate a 60–70 nt stem loop structure known as precursor miRNA (pre-miRNA) [3]. The pre-miRNA is then exported to the cytoplasm by an Exportin 5-dependent mechanism and further processed into a transient ∼22 bp miRNA:miRNA duplex ( indicates the passenger strand, which is not complementary to the RNA target) again by DCL1 in plants [3]. The miRNA:miRNA duplex is then loaded into the RNA-induced silencing complex (RISC). Only the mature miRNA strand is retained in the complex, while the miRNA is degraded. The mature miRNA is used as a template to guide the silencing of complementary target mRNAs [5]. In plants, miRNAs display a high degree of complementary with their targets and produce silencing mostly through mRNA cleavage, however translational repression has also been documented [6].

Section snippets

Why amiRNAs were needed, design and advantages?

Prior to the development of amiRNAs, various strategies exploited the PTGS pathway to produce gene knockouts and study gene function. These strategies were mainly based on the production of siRNAs derived from dsRNAs introduced to the plant in various ways. One strategy known as virus-induced gene silencing which employs viral genomes as vectors to introduce dsRNAs to silence any desired gene in the plant to study the phenotype produced [7]. Although the approach has certain advantages, the

Arabidopsis thaliana

Since early in their development in molecular biology model plant A. thaliana, amiRNAs were found to exert efficient silencing in plants and their advantages in terms of specificity, effectiveness have increased with research over the last few years [13], [14], [22]. As an indication of the potential of amiRNAs in Arabidopsis research, a library of amiRNAs was developed and is currently kept at Cold Spring Harbor Laboratories, where each of the estimated 22,000 A. thaliana genes are targeted by

amiRNAs and potential applications in plant immunity

amiRNAs also hold a potential great impact in plant pathology, mainly, the use of amiRNAs to engineer plants resistant to viruses has been extensively explored [10], [18]. In an early work, amiRNAs were designed (based on an A. thaliana miR159 precursor) to target viral mRNA sequences coding two silencing suppressors: P69 of the turnip yellow mosaic virus (TYMV) and HC-Pro of the turnip mosaic virus (TuMV). Transgenic A. thaliana plants expressing amiR-P69 and amiR-HC-Pro were specifically

amiRNAs advances in crops research

There is currently a lot of ongoing work using amiRNAs as the tool of choice for functional genetic studies, and they are spreading rapidly to important crops and to other plants. In wheat, for example, amiRNAs were used to silence the 5-methylcytosine glycosylase gene responsible for the endosperm-specific demethylation and transcriptional activation, named DEMETER. amiRNAs were specifically expressed under the control of an endosperm-specific promoter to eliminate low molecular weight

amiRNA as effective endogenous miRNA regulator

Interestingly, amiRNAs where recently used to silence endogenous miRNAs in Arabidopsis[45]. amiRNAs where specifically designed to target miRNA families miR159 and miR164, which target the GAMYB-like family of transcription factors and the CUC and NAC transcription factors, respectively. These families are expressed by multiple loci and the mature protein shows high sequence conservation. The amiRNA-mediated silencing was exerted over all members of each family [45]. The transformed plants

Acknowledgments

Gaurav Sablok thanks Key Lab of Horticultural Plant Biology (MOE), Huazhong Agricultural University. Tatiana Tatarinova would like to thank the University of Glamorgan’s Research Investment Scheme for supporting this project. Financial support for Camilo López and Alvaro Perez comes from Dirección de Investigaciones sede Bogota (Universidad Nacional), Colciencias, and Ministerio de Agricultura de Colombia.

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