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Comparative Study
. 2005 Aug 23;102(34):12023-8.
doi: 10.1073/pnas.0500053102. Epub 2005 Aug 12.

Targets of microRNA regulation in the Drosophila oocyte proteome

Kenji Nakahara  1 , Kevin Kim, Christin Sciulli, Susan R Dowd, Jonathan S Minden, Richard W Carthew
Affiliations
Comparative Study

Targets of microRNA regulation in the Drosophila oocyte proteome

Kenji Nakahara et al. Proc Natl Acad Sci U S A. .

Abstract

MicroRNAs (miRNAs) are a class of small RNAs that silence gene expression. In animal cells, miRNAs bind to the 3' untranslated regions of specific mRNAs and inhibit their translation. Although some targets of a handful of miRNAs are known, the number and identities of mRNA targets in the genome are uncertain, as are the developmental functions of miRNA regulation. To identify the global range of miRNA-regulated genes during oocyte maturation of Drosophila, we compared the proteome from wild-type oocytes with the proteome from oocytes lacking the dicer-1 gene, which is essential for biogenesis of miRNAs. Most identified proteins appeared to be subject to translation inhibition. Their transcripts contained putative binding sites in the 3' untranslated region for a subset of miRNAs, based on computer modeling. The fraction of genes subject to direct and indirect repression by miRNAs during oocyte maturation appears to be small (4%), and the genes tend to share a common functional relationship in protein biogenesis and turnover. The preponderance of genes that control global protein abundance suggests this process is under tight control by miRNAs at the onset of fertilization.

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Figures

Fig. 1.
Fig. 1.
DIGE analysis of dcr-1 eggs. (A) Dorsal view of mature wild-type (Left) and dcr-1 mutant oocytes (Right). Anterior is down. The mutant oocyte is reduced in size and has a partial fusion of dorsal appendages, indicating a ventralized phenotype. (B) Schematic of DIGE. In this example, proteins from dcr-1 mutants are coupled to Cy3 and wild-type are coupled to Cy5. Cy3 fluorescence is imaged purple and Cy5 is imaged green. Protein spots that fluoresce equally with both dyes appear white. Spots that fluoresce more intensely from one dye than the other appear either purple or green. These represent difference proteins. (C) A 2D gel scanned for Cy5-tagged proteins that had been isolated from dcr-1 mutants. Circled are the 41 protein spots, referred to as dcr-1-enriched proteins, which are reproducibly more fluorescent for proteins from the mutant than wild-type. Numbers refer to spot identities as listed in Table 1. (D) Magnified view of a portion of a scanned 2D gel showing protein spots containing dcr-1 mutant proteins tagged with Cy3 (purple) and wild-type proteins tagged with Cy5 (green). Spots that exhibit greater Cy3 fluorescence appear purple, and those spots that reproducibly exhibited greater Cy3 fluorescence are numbered. Some of the spots that appear purple in this gel were not reproducible and so are not numbered. Numbers refer to spot identities as listed in Table 1. (E) Ratio of fluorescence from dcr-1 to wild-type spot samples normalized to the background variation. The ratio is expressed as the number of standard deviations above the background mean.
Fig. 2.
Fig. 2.
Levels of mRNA transcripts in wild-type (black bars) or dcr-1 mutant (white bars) oocytes. Shown are levels of transcripts from the indicated genes, normalized to the corresponding levels of rp49 transcript in each sample, and determined by semiquantitative RT-PCR. For each gene, the level from the wild-type sample is set to a value of 1.0. The data displayed are from one experiment, and transcript data listed in Table 2 represent averages of values from replicate experiments.
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