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Principles of microRNA-target recognition.

Brennecke J, Stark A, Russell RB, Cohen SM - PLoS Biol. (2005)

Bottom Line: In contrast, 3' compensatory sites have insufficient 5' pairing and require strong 3' pairing for function.We present examples and genome-wide statistical support to show that both classes of sites are used in biologically relevant genes.We provide evidence that an average miRNA has approximately 100 target sites, indicating that miRNAs regulate a large fraction of protein-coding genes and that miRNA 3' ends are key determinants of target specificity within miRNA families.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Heidelberg, Germany.

ABSTRACT
MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression in plants and animals. Although their biological importance has become clear, how they recognize and regulate target genes remains less well understood. Here, we systematically evaluate the minimal requirements for functional miRNA-target duplexes in vivo and distinguish classes of target sites with different functional properties. Target sites can be grouped into two broad categories. 5' dominant sites have sufficient complementarity to the miRNA 5' end to function with little or no support from pairing to the miRNA 3' end. Indeed, sites with 3' pairing below the random noise level are functional given a strong 5' end. In contrast, 3' compensatory sites have insufficient 5' pairing and require strong 3' pairing for function. We present examples and genome-wide statistical support to show that both classes of sites are used in biologically relevant genes. We provide evidence that an average miRNA has approximately 100 target sites, indicating that miRNAs regulate a large fraction of protein-coding genes and that miRNA 3' ends are key determinants of target specificity within miRNA families.

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Complementarity to the miRNA 5′ End Is Important for Target Site Function In Vivo(A) In vivo assay for target site regulation in the wing imaginal disc. The EGFP reporter is expressed in all cells (green). Cells expressing the miRNA under ptcGal4 control are shown in red. Functional target sites allow strong GFP repression by the miRNA (middle). Non-functional target sites do not (right). Yellow boxes indicate the disc region shown in (B) and later figures.(B) Regulation of individual target sites by miR-7. Numbers in the upper left of each image indicate the mismatched nucleotide in the target site. Positions important for regulation are shown in red, dispensable positions in green. Regulation by the miRNA is completely abolished in only a few cases.(C) Summary of the magnitude of reporter gene repression for the series in (B) and for a second set involving miR-278 and a target site resembling the miR-9 site in Lyra [26]. Positions important for regulation are shown in red, dispensable positions in green. Error bars are based on measurements of 3–5 individual discs.
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pbio-0030085-g001: Complementarity to the miRNA 5′ End Is Important for Target Site Function In Vivo(A) In vivo assay for target site regulation in the wing imaginal disc. The EGFP reporter is expressed in all cells (green). Cells expressing the miRNA under ptcGal4 control are shown in red. Functional target sites allow strong GFP repression by the miRNA (middle). Non-functional target sites do not (right). Yellow boxes indicate the disc region shown in (B) and later figures.(B) Regulation of individual target sites by miR-7. Numbers in the upper left of each image indicate the mismatched nucleotide in the target site. Positions important for regulation are shown in red, dispensable positions in green. Regulation by the miRNA is completely abolished in only a few cases.(C) Summary of the magnitude of reporter gene repression for the series in (B) and for a second set involving miR-278 and a target site resembling the miR-9 site in Lyra [26]. Positions important for regulation are shown in red, dispensable positions in green. Error bars are based on measurements of 3–5 individual discs.

Mentions: To improve our understanding of the minimal requirements for a functional miRNA target site, we made use of a simple in vivo assay in the Drosophila wing imaginal disc. We expressed a miRNA in a stripe of cells in the central region of the disc and assessed its ability to repress the expression of a ubiquitously transcribed enhanced green fluorescent protein (EGFP) transgene containing a single target site in its 3′ UTR. The degree of repression was evaluated by comparing EGFP levels in miRNA-expressing and adjacent non-expressing cells. Expression of the miRNA strongly reduced EGFP expression from transgenes containing a single functional target site (Figure 1A).


Principles of microRNA-target recognition.

Brennecke J, Stark A, Russell RB, Cohen SM - PLoS Biol. (2005)

Complementarity to the miRNA 5′ End Is Important for Target Site Function In Vivo(A) In vivo assay for target site regulation in the wing imaginal disc. The EGFP reporter is expressed in all cells (green). Cells expressing the miRNA under ptcGal4 control are shown in red. Functional target sites allow strong GFP repression by the miRNA (middle). Non-functional target sites do not (right). Yellow boxes indicate the disc region shown in (B) and later figures.(B) Regulation of individual target sites by miR-7. Numbers in the upper left of each image indicate the mismatched nucleotide in the target site. Positions important for regulation are shown in red, dispensable positions in green. Regulation by the miRNA is completely abolished in only a few cases.(C) Summary of the magnitude of reporter gene repression for the series in (B) and for a second set involving miR-278 and a target site resembling the miR-9 site in Lyra [26]. Positions important for regulation are shown in red, dispensable positions in green. Error bars are based on measurements of 3–5 individual discs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1043860&req=5

pbio-0030085-g001: Complementarity to the miRNA 5′ End Is Important for Target Site Function In Vivo(A) In vivo assay for target site regulation in the wing imaginal disc. The EGFP reporter is expressed in all cells (green). Cells expressing the miRNA under ptcGal4 control are shown in red. Functional target sites allow strong GFP repression by the miRNA (middle). Non-functional target sites do not (right). Yellow boxes indicate the disc region shown in (B) and later figures.(B) Regulation of individual target sites by miR-7. Numbers in the upper left of each image indicate the mismatched nucleotide in the target site. Positions important for regulation are shown in red, dispensable positions in green. Regulation by the miRNA is completely abolished in only a few cases.(C) Summary of the magnitude of reporter gene repression for the series in (B) and for a second set involving miR-278 and a target site resembling the miR-9 site in Lyra [26]. Positions important for regulation are shown in red, dispensable positions in green. Error bars are based on measurements of 3–5 individual discs.
Mentions: To improve our understanding of the minimal requirements for a functional miRNA target site, we made use of a simple in vivo assay in the Drosophila wing imaginal disc. We expressed a miRNA in a stripe of cells in the central region of the disc and assessed its ability to repress the expression of a ubiquitously transcribed enhanced green fluorescent protein (EGFP) transgene containing a single target site in its 3′ UTR. The degree of repression was evaluated by comparing EGFP levels in miRNA-expressing and adjacent non-expressing cells. Expression of the miRNA strongly reduced EGFP expression from transgenes containing a single functional target site (Figure 1A).

Bottom Line: In contrast, 3' compensatory sites have insufficient 5' pairing and require strong 3' pairing for function.We present examples and genome-wide statistical support to show that both classes of sites are used in biologically relevant genes.We provide evidence that an average miRNA has approximately 100 target sites, indicating that miRNAs regulate a large fraction of protein-coding genes and that miRNA 3' ends are key determinants of target specificity within miRNA families.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Heidelberg, Germany.

ABSTRACT
MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression in plants and animals. Although their biological importance has become clear, how they recognize and regulate target genes remains less well understood. Here, we systematically evaluate the minimal requirements for functional miRNA-target duplexes in vivo and distinguish classes of target sites with different functional properties. Target sites can be grouped into two broad categories. 5' dominant sites have sufficient complementarity to the miRNA 5' end to function with little or no support from pairing to the miRNA 3' end. Indeed, sites with 3' pairing below the random noise level are functional given a strong 5' end. In contrast, 3' compensatory sites have insufficient 5' pairing and require strong 3' pairing for function. We present examples and genome-wide statistical support to show that both classes of sites are used in biologically relevant genes. We provide evidence that an average miRNA has approximately 100 target sites, indicating that miRNAs regulate a large fraction of protein-coding genes and that miRNA 3' ends are key determinants of target specificity within miRNA families.

Show MeSH