Abstract
BackgroundRecently, putative pre-miRNAs locations have been identified in the introns of plant genes, raising the question whether such genes can show a dual functionality by having both correct maturation of the host gene pre-mRNA and maturation of the miRNAs from the intron. Here, we demonstrated that such dual functionality is indeed possible, using as host gene the firefly luciferase gene with intron (ffgLUC), and different artificial intronic miRNAs (aimiRNA) placed within the intron of ffgLUC.ResultsThe miRNAs were based on the structure of the natural miR319a. Luciferase (LUC) activity in planta was used to evaluate a correct splicing of the ffgLUC mRNA. Different target sequences were inserted into the aimiRNA to monitor efficiency of silencing of different target mRNAs. After adjusting the insertion cloning strategy, the ffgLUCaimiR-319a gene showed dual functionality with correct splicing of ffgLUC and efficient silencing of TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 transcription factor genes targeted in-trans by aimiR-319a or targeting the transgene ffLUC in-cis by an aimiR-LUC. Silencing of endogenous target genes by aimiRNA or amiRNA is efficient both in transient assays and stable transformants. A behave as strong phenotype the PHYTOCHROME B (PHYB) gene was also targeted by ffgLUCaimiR-PHYB. The lack of silencing of the PHYB target was most likely due to an insensitive target site within the PHYB mRNA which can potentially form a double stranded stem structure.ConclusionThe combination of an overexpression construct with an artificial intronic microRNA allows for a simultaneous dual function in plants. The concept therefore adds new options to engineering of plant traits that require multiple gene manipulations.
Highlights
Putative pre-miRNAs locations have been identified in the introns of plant genes, raising the question whether such genes can show a dual functionality by having both correct maturation of the host gene premRNA and maturation of the miRNAs from the intron
We tested whether endogenous N. benthamiana TCP4 (NbTCP4) gene expression was affected by ffgLUCdaieml iR-319a as the AthTCP target sequence of aimiR-319a shows substantial overlap with
It could be that the reduced distance between 5′-splice site and Intronic microRNA (imiRNA) sequence in ffgLUCdaieml iR-319a resulted in spatial constraints because of simultaneous assembly of spliceosome and miRNA-processing protein complexes
Summary
Putative pre-miRNAs locations have been identified in the introns of plant genes, raising the question whether such genes can show a dual functionality by having both correct maturation of the host gene premRNA and maturation of the miRNAs from the intron. Important traits of crop plants have successfully been manipulated by selection of mutants [1], by ectopic expression of a transgene [2,3,4], or by silencing of a single gene [5, 6]. Stacking of MicroRNAs (miRNA) are short (19-22nt) non coding RNAs that can silence the expression of specific target genes and natural miRNAs form an integral part of developmental decisions in plants [9, 10]. While most miRNA are processed from regular non-coding miRNA-genes, recently, protein-coding genes with introns containing potential miRNA sequences have been identified both in mammals
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