miRNA-Induced Gene Silencing (MIGS): for regulation of Gene Expression in Crop Plants
Basavaprabhu L. Patil*, Rohini Sreevathsa and Monika Dalal
National Research Centre on Plant Biotechnology, Pusa, New Delhi-110012
*Corresponding author e-mail: firstname.lastname@example.org
Gene silencing is one of the most important mechanisms of gene regulation in plants, which is mediated by 19-24 nt long RNA molecules, also known as small RNAs (sRNAs). The process of gene silencing, which is also referred as RNA interference (RNAi), regulates gene expression, either by post-transcriptional gene silencing (PTGS) or by transcriptional gene silencing (TGS). In plants, the small RNAs can be grouped into two major classes, microRNAs (miRNAs) and small interfering RNAs (siRNAs). In both the cases, the small RNAs are produced from double-stranded RNAs (dsRNA) precursors through the action of Dicer-Like (DCL) enzymes. The resulting small RNAs are then loaded onto the Argonaute (AGO) proteins to form the RNA-Induced Silencing Complex (RISC). Subsequently the RISC complex will result in downregulation of the gene expression either by cleavage of the RNA transcript (PTGS) or by DNA methylation (TGS).
Trans-acting siRNAs (tasiRNAs) are recently identified class of small RNAs, which are derived from TAS gene-derived transcripts after being acted upon by a miRNA. The miRNA-dependent cleavage of the TAS transcript results in the recruitment of SGS3 (Suppressor of Gene Silencing 3) and RDR6 (RNA-Dependent RNA Polymerase 6). The RDR6 uses the TAS transcript as a template for the synthesis of dsRNA, which is processed by DCL4 (DICER-LIKE 4) into tasiRNAs of 21nts size and are subsequently loaded onto AGO1 (Argonaute 1) and eventually promote the downregulation of the genes in trans. In case of Arabidopsis, the model plant, four TAS families have been identified. The miRNA173 directs the cleavage of TAS1 and TAS2 leading to the generation of tasiRNAs from the sequences located downstream of miRNA173 recognition site. The requirements for the generation of tasiRNAs is not fully understood; however studies have shown that only those miRNAs which are of 22 nt in size are capable of generation of tasiRNAs and not those which are 21 nt in size. In case of Arabidopsis, miR173 and miR390 are identified to be of 22 nt in size, otherwise most of the miRNAs are 21 nt in size. The cleavage mediated by miR173 is sufficient to initiate transitivity, and targeting of a given gene by miR173 results in the production of secondary siRNAs originating from the target nucleotide sequence.
The above studies lead to the emergence of a gene regulation technique termed as "miRNA-Induced Gene Silencing" (MIGS). It is essentially based on the unique feature of the miR173 to trigger the generation of secondary siRNAs (tasiRNAs) from its target sequences. The concept behind MIGS is simple; it mainly involves targeting the miR173 to a gene sequence, for which silencing/downregulation is desired. This is achieved by addition of the miRNA target site in the immediate upstream of the sequence of interest. The construction of MIGS vectors is simple and does not involve multiple steps of PCR amplification and/or cloning. To facilitate the use of MIGS technology, a large collection of MIGS plasmids based on the pGreen binary vector have been generated by Dr. Weigel of Max Planck Institute for Developmental Biology in Tubingen (Germany). These vectors are Gateway compatible, hence enable high throughput cloning and confer either BASTA or kanamycin resistance in plants, which can be conveniently used for selection of the transgenic plants. When the transcript produced by the MIGS construct (the target sequence flanked by miR173 target site) is cleaved by the miR173, the tasiRNA generation initiates, resulting in the production of secondary siRNAs. However, miR390 is not suitable for its incorporation in the MIGS vector, since it specifically associates with AGO7, and the miR390-based MIGS would be limited to the site of AGO7 expression, which is restricted to the plant vascular system. MIGS can also be used to simultaneously silence multiple genes by fusing multiple MIGS modules (miR173 target site plus the sequence of interest) to generate a single MIGS construct, which subsequently can be then cloned into a binary vector of choice for plant transformation. This fusion MIGS construct is capable of simultaneously silencing different genes with same efficiency.
The MIGS technology offers several advantages over other techniques of inducing gene silencing (hpRNA or amiRNA):
1. MIGS vectors are easily generated after a single step of PCR.
2. In the case of hpRNAi (hairpin RNAi), a fragment of the gene of interest needs to be cloned as an inverted repeat, along with the intron and this is not required for MIGS. This significantly reduces the size of the insert and enables stacking of multiple sequences.
3. Design of amiRNAs (artificial micro RNAs) often requires multiple PCR steps for replacement of the mature miRNA in the precursor backbone.
4. Ability to silence multiple unrelated genes using a single MIGS vector.
However some of the concerns associated with the MIGS technology are similar to those that apply to hpRNA and VIGS. In some of the plant species the tasiRNA trigger miR173 is not expressed endogenously and in such cases it needs to be co-expressed as an amiRNA. These strategies can be incorporated in crop improvement programmes for the successful mitigation of various biotic and abiotic stresses, provided there is adequate knowledge of the target sequences.
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About Author / Additional Info:
All the authors are Senior Scientists (Agricultural Biotechnology) at National Research Center on Plant Biotechnology (NRCPB), New Delhi.