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A collection of target mimics for comprehensive analysis of microRNA function in Arabidopsis thaliana.

Todesco M, Rubio-Somoza I, Paz-Ares J, Weigel D - PLoS Genet. (2010)

Bottom Line: Morphological defects in the aerial part were observed for approximately 20% of analyzed families, all of which are deeply conserved in land plants.That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development.In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany.

ABSTRACT
Many targets of plant microRNAs (miRNAs) are thought to play important roles in plant physiology and development. However, because plant miRNAs are typically encoded by medium-size gene families, it has often been difficult to assess their precise function. We report the generation of a large-scale collection of knockdowns for Arabidopsis thaliana miRNA families; this has been achieved using artificial miRNA target mimics, a recently developed technique fashioned on an endogenous mechanism of miRNA regulation. Morphological defects in the aerial part were observed for approximately 20% of analyzed families, all of which are deeply conserved in land plants. In addition, we find that non-cleavable mimic sites can confer translational regulation in cis. Phenotypes of plants expressing target mimics directed against miRNAs involved in development were in several cases consistent with previous reports on plants expressing miRNA-resistant forms of individual target genes, indicating that a limited number of targets mediates most effects of these miRNAs. That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development. In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.

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Effects of artificial mimics on levels of miRNAs and miRNA targets.(A) Nine-day-old plants. Introduction of a MIM159 fragment into the 3′ UTR silences a constitutively expressed 3xEYFP in the MIR159 expression domain (compare p35S:3xEYFP and p35S:3xEYFP-MIM159), which is revealed in the pMIR159:GUS lines. MiR159 activity is also indirectly revealed by comparing the effect of expressing MIM159 in a genomic MYB33:GUS line. (B) Transcript levels of select miRNA targets in two independent lines for each MIM construct (represented by bars of different shades of gray). (C) Expression levels of miRNA targets in mutants impaired in miRNA biogenesis or targeting. Expression values are reported as the average of two biological and two technical replicates, and are normalized to the expression levels in wild type Col-0 plants (dotted line). (D) CIP4 mRNA and protein levels in four independent MIM834 lines. Band intensity relative to the wild-type control is reported. (E) Levels of mature miRNAs in several MIM lines. U6 accumulation is shown as control. Increased accumulation of miR156 (lower band in the blot) was observed upon expression of a resistant version of a miR156 target (consistent with what observed for miRNA156a precursor levels in [39]) or inhibition of miRNA activity in the ago1-27 mutants. The decrease in miR156 levels in MIM156 plants is then not an indirect consequence of increased SPL transcript levels.
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pgen-1001031-g004: Effects of artificial mimics on levels of miRNAs and miRNA targets.(A) Nine-day-old plants. Introduction of a MIM159 fragment into the 3′ UTR silences a constitutively expressed 3xEYFP in the MIR159 expression domain (compare p35S:3xEYFP and p35S:3xEYFP-MIM159), which is revealed in the pMIR159:GUS lines. MiR159 activity is also indirectly revealed by comparing the effect of expressing MIM159 in a genomic MYB33:GUS line. (B) Transcript levels of select miRNA targets in two independent lines for each MIM construct (represented by bars of different shades of gray). (C) Expression levels of miRNA targets in mutants impaired in miRNA biogenesis or targeting. Expression values are reported as the average of two biological and two technical replicates, and are normalized to the expression levels in wild type Col-0 plants (dotted line). (D) CIP4 mRNA and protein levels in four independent MIM834 lines. Band intensity relative to the wild-type control is reported. (E) Levels of mature miRNAs in several MIM lines. U6 accumulation is shown as control. Increased accumulation of miR156 (lower band in the blot) was observed upon expression of a resistant version of a miR156 target (consistent with what observed for miRNA156a precursor levels in [39]) or inhibition of miRNA activity in the ago1-27 mutants. The decrease in miR156 levels in MIM156 plants is then not an indirect consequence of increased SPL transcript levels.

Mentions: Artificial target mimics are thought to sequester their target miRNAs, presumably by stably binding to miRNA-loaded RISCs. To obtain additional evidence for such interactions, we embedded a functional MIM159 site in the 3′-UTR of a triple- Enhanced Yellow Fluorescent Protein (EYFP) reporter; stable recruitment of RISCmiR399 to the mimic site could be expected to interfere with EYFP translation. In 80% of MIM159 expressing T1 plants, as in control plants, the EYFP transgene was completely silenced. In the remaining 20%, we detected EYFP signal that was strongly reduced in the region where MIR159 genes are known to be expressed (Figure 4A) [26]. In addition, these plants presented the typical phenotypic defects of MIM159 plants, confirming that the EYFP:MIM159 construct functions properly as a target mimic.


A collection of target mimics for comprehensive analysis of microRNA function in Arabidopsis thaliana.

Todesco M, Rubio-Somoza I, Paz-Ares J, Weigel D - PLoS Genet. (2010)

Effects of artificial mimics on levels of miRNAs and miRNA targets.(A) Nine-day-old plants. Introduction of a MIM159 fragment into the 3′ UTR silences a constitutively expressed 3xEYFP in the MIR159 expression domain (compare p35S:3xEYFP and p35S:3xEYFP-MIM159), which is revealed in the pMIR159:GUS lines. MiR159 activity is also indirectly revealed by comparing the effect of expressing MIM159 in a genomic MYB33:GUS line. (B) Transcript levels of select miRNA targets in two independent lines for each MIM construct (represented by bars of different shades of gray). (C) Expression levels of miRNA targets in mutants impaired in miRNA biogenesis or targeting. Expression values are reported as the average of two biological and two technical replicates, and are normalized to the expression levels in wild type Col-0 plants (dotted line). (D) CIP4 mRNA and protein levels in four independent MIM834 lines. Band intensity relative to the wild-type control is reported. (E) Levels of mature miRNAs in several MIM lines. U6 accumulation is shown as control. Increased accumulation of miR156 (lower band in the blot) was observed upon expression of a resistant version of a miR156 target (consistent with what observed for miRNA156a precursor levels in [39]) or inhibition of miRNA activity in the ago1-27 mutants. The decrease in miR156 levels in MIM156 plants is then not an indirect consequence of increased SPL transcript levels.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908682&req=5

pgen-1001031-g004: Effects of artificial mimics on levels of miRNAs and miRNA targets.(A) Nine-day-old plants. Introduction of a MIM159 fragment into the 3′ UTR silences a constitutively expressed 3xEYFP in the MIR159 expression domain (compare p35S:3xEYFP and p35S:3xEYFP-MIM159), which is revealed in the pMIR159:GUS lines. MiR159 activity is also indirectly revealed by comparing the effect of expressing MIM159 in a genomic MYB33:GUS line. (B) Transcript levels of select miRNA targets in two independent lines for each MIM construct (represented by bars of different shades of gray). (C) Expression levels of miRNA targets in mutants impaired in miRNA biogenesis or targeting. Expression values are reported as the average of two biological and two technical replicates, and are normalized to the expression levels in wild type Col-0 plants (dotted line). (D) CIP4 mRNA and protein levels in four independent MIM834 lines. Band intensity relative to the wild-type control is reported. (E) Levels of mature miRNAs in several MIM lines. U6 accumulation is shown as control. Increased accumulation of miR156 (lower band in the blot) was observed upon expression of a resistant version of a miR156 target (consistent with what observed for miRNA156a precursor levels in [39]) or inhibition of miRNA activity in the ago1-27 mutants. The decrease in miR156 levels in MIM156 plants is then not an indirect consequence of increased SPL transcript levels.
Mentions: Artificial target mimics are thought to sequester their target miRNAs, presumably by stably binding to miRNA-loaded RISCs. To obtain additional evidence for such interactions, we embedded a functional MIM159 site in the 3′-UTR of a triple- Enhanced Yellow Fluorescent Protein (EYFP) reporter; stable recruitment of RISCmiR399 to the mimic site could be expected to interfere with EYFP translation. In 80% of MIM159 expressing T1 plants, as in control plants, the EYFP transgene was completely silenced. In the remaining 20%, we detected EYFP signal that was strongly reduced in the region where MIR159 genes are known to be expressed (Figure 4A) [26]. In addition, these plants presented the typical phenotypic defects of MIM159 plants, confirming that the EYFP:MIM159 construct functions properly as a target mimic.

Bottom Line: Morphological defects in the aerial part were observed for approximately 20% of analyzed families, all of which are deeply conserved in land plants.That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development.In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany.

ABSTRACT
Many targets of plant microRNAs (miRNAs) are thought to play important roles in plant physiology and development. However, because plant miRNAs are typically encoded by medium-size gene families, it has often been difficult to assess their precise function. We report the generation of a large-scale collection of knockdowns for Arabidopsis thaliana miRNA families; this has been achieved using artificial miRNA target mimics, a recently developed technique fashioned on an endogenous mechanism of miRNA regulation. Morphological defects in the aerial part were observed for approximately 20% of analyzed families, all of which are deeply conserved in land plants. In addition, we find that non-cleavable mimic sites can confer translational regulation in cis. Phenotypes of plants expressing target mimics directed against miRNAs involved in development were in several cases consistent with previous reports on plants expressing miRNA-resistant forms of individual target genes, indicating that a limited number of targets mediates most effects of these miRNAs. That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development. In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.

Show MeSH
Related in: MedlinePlus