Targets of Microrna Regulation in the Drosophila Oocyte Proteome

Targets of microRNA regulation in the Drosophila oocyte proteome

Kenji Nakahara*†‡, Kevin Kim*†, Christin Sciulli§, Susan R. Dowd§, Jonathan S. Minden§, and Richard W. Carthew*¶

*Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208; and §Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213

Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved July 6, 2005 (received for review January 22, 2005) MicroRNAs (miRNAs) are a class of small RNAs that silence gene differentiation (11). In Drosophila, translation of the apoptosis .(expression. In animal cells, miRNAs bind to the 3؅ untranslated effector gene hid is down-regulated by the bantam miRNA (12 regions of specific mRNAs and inhibit their translation. Although A different approach to finding gene targets of miRNAs has some targets of a handful of miRNAs are known, the number and been to scan sequence databases for conserved sequences within identities of mRNA targets in the genome are uncertain, as are the 3Ј UTRs that are favored to interact with miRNAs (13–18). developmental functions of miRNA regulation. To identify the Several predicted targets have been experimentally validated, global range of miRNA-regulated genes during oocyte maturation suggesting that these computational methods can predict ge- of Drosophila, we compared the proteome from wild-type oocytes nome-wide targets. However, it is unclear how well these theo- with the proteome from oocytes lacking the dicer-1 gene, which is retical analyses identify genes that are directly regulated by essential for biogenesis of miRNAs. Most identified proteins ap- miRNAs. peared to be subject to translation inhibition. Their transcripts In this study, we have undertaken a proteomic screen for genes contained putative binding sites in the 3؅ untranslated region for that are regulated by miRNAs within late-stage Drosophila a subset of miRNAs, based on computer modeling. The fraction of oocytes. We find a small percentage (Ϸ4%) of expressed genes genes subject to direct and indirect repression by miRNAs during are derepressed when miRNA function is lost. Identification of oocyte maturation appears to be small (4%), and the genes tend to those genes by MS indicates that many of these genes function share a common functional relationship in protein biogenesis and in protein biogenesis and turnover. Thus, miRNAs selectively turnover. The preponderance of genes that control global protein regulate related functional groups of genes at this stage of abundance suggests this process is under tight control by miRNAs development. at the onset of fertilization. Materials and Methods translation control Genetics. To generate null dicer-1 (dcr-1) oocytes, FRT82B dcr- 1Q1147X͞TM6B females were crossed with hsFLP122;FRT82B ovoD1͞TM6B males, and progeny were heat-shocked twice at mall RNAs, including microRNAs (miRNAs) and short 37°C for 2 h during early larval stages, as described (19). F interfering RNAs, are components of a RNA-based mech- 1 S females were induced to lay mature oocytes that were deposited anism of gene silencing (1, 2). The miRNA branch of RNA-based onto egg-laying plates. Wild-type oocytes were collected from gene regulation is found in plants and animals (3, 4). miRNAs ͞ D1 Ϸ hsFLP122;FRT82B FRT82B ovo females treated in the same have a specific size of 22 nt, and they are processed from manner as above. Oocytes were collected, pooled in batches of hairpin-loop RNA precursors by endoribonucleases of the Dicer 200, and frozen as described (20). class. These precursors are transcribed from genes within plant and animal genomes, with the number of miRNA genes found Difference Gel Electrophoresis (DIGE) and MS. Seventy to 100 ␮gof in different species roughly corresponding to 0.5–1% of the total protein homogenate from a particular genotype was conjugated number of genes in their genomes (5). Most miRNA genes are Ϸ with either Cy3 or Cy5 dye, mixed with equal protein from the conserved between related species, and 30% of miRNA genes other genotype that had been coupled with the opposite dye, and are highly conserved, with orthologs found in vertebrate and the mixture subjected to DIGE analysis, as described (20). invertebrate genomes. This suggests that a significant fraction of Positive protein spots were excised from five gels, and proteins miRNAs have evolutionarily conserved biological functions. were extracted, digested with trypsin, and analyzed by a Per- miRNAs specifically repress gene expression by negatively Spective Biosystems Voyager STR MALDI-TOF, as described regulating complementary mRNAs. Plant miRNAs generally (20). Data were analyzed by using MASCOT (21). Details of DIGE cause degradation of complementary mRNAs by near-perfect analysis and imaging are in Supporting Text, which is published basepairing (5). Conversely, most characterized miRNAs from as supporting information on the PNAS web site. animals repress gene expression by blocking the translation of complementary mRNAs into protein. They interact with their RT-PCR. RNA was isolated from dcr-1Q1147X and wild-type oo- targets by imperfectly basepairing to mRNA sequences within cytes, and cDNA was generated as described (22). The primers the 3Ј UTR. The exact mechanism of translation inhibition is used to amplify ribosomal protein (rp)49 cDNA were described unknown, although miRNAs have been found not to interfere (23), and primers positioned in the 3Ј UTR of each positive with translation initiation (5). target gene were used to amplify their cDNAs (see Supporting A question of great interest concerns the functions of miRNAs in animals. Although the functions of only a few miRNAs are known, available evidence suggests that miRNAs play diverse This paper was submitted directly (Track II) to the PNAS office. and important roles in development. This conclusion is based on Abbreviations: miRNA, microRNA; DIGE, difference gel electrophoresis; dcr-1, dicer-1; rp, the mutant phenotypes of individual miRNA genes and on the ribosomal protein. identification of genes that are direct targets of miRNA regu- †K.N. and K.K. contributed equally to this work. lation. In Caenorhabditis elegans, the genes lin-14, lin-28, lin-41, ‡Present address: Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, and hbl-1 are translationally repressed by interaction with lin-4 Japan.

or let-7 miRNAs (6–10). Neuronal development is regulated by ¶To whom correspondence should be addressed. E-mail: [email protected]. BIOCHEMISTRY sequential action of lsy-6 and miR-273 on effectors of neuron © 2005 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500053102 PNAS ͉ August 23, 2005 ͉ vol. 102 ͉ no. 34 ͉ 12023–12028 Downloaded by guest on September 29, 2021 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.

Primer Data Set, which is published as supporting information on An important issue in proteomic analysis of any mutant is the the PNAS web site, for primer sequences). Each reaction was possible impact of the mutation on cell composition at any given performed as described (23). Signals were normalized to the stage of development. For example, mutant dcr-1 oocytes are control rp49 levels in each sample. ventralized (Fig. 1A), which make it likely that resulting embryos develop with a greater proportion of ventral cell types such as 3؅ UTR Sequence Analysis. The annotated 3Ј UTR sequence for neurons and muscle. The proteome profile of such embryos each gene was collated as described (14). Details of algorithms, would reflect this altered cell composition in addition to proteins sequences, and statistical analysis are given in Supporting Text. that might be autonomously controlled by miRNAs. For this reason, we chose to perform our analysis on mature oocytes. Results These constitute a homogeneous cell population, which reduces We performed comparative proteomics to identify genes that are the likelihood that any proteome differences are due to heter- regulated by miRNAs. We assumed that the protein products of ogeneity in cell composition (25). Moreover, dcr-1 mutant such genes would be more abundant in the absence of mature oocytes could be fertilized by wild-type sperm and undergo miRNAs. To identify this population of proteins, we used DIGE embryonic development, demonstrating that mutant oocytes (20, 24) and compared the proteome of normal individuals with reached this developmental stage (data not shown). the proteome of those depleted of miRNAs. It was not possible To compare proteomes of wild-type and dcr-1 individuals, we to genetically ablate all miRNAs from the genome, given their covalently labeled protein homogenates with Cy3 or Cy5 dyes number. Therefore, we examined the proteome of dcr-1 null that did not significantly alter protein charge or mass (Fig. 1B). mutants. The dcr-1 gene encodes for the Dicer enzyme essential The Cy3- and Cy5-labeled homogenates were combined and for miRNA biogenesis in Drosophila (22). A dcr-1 null mutant is electrophoresed on the same 2D gel. Fluorescence imaging of the blocked for miRNA processing, leading to an absence of mature gel with Cy3 and Cy5 excitation light generated images of the two miRNAs. The mutant, however, is not defective for short protein samples in perfect register. Proteins common to both interfering RNA processing, because another Dicer gene carries samples appeared as spots that fluoresced with both dyes. out this role (22). Proteins that were more abundant in one sample appeared as

12024 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500053102 Nakahara et al. Downloaded by guest on September 29, 2021 spots that fluoresced more intensely with one dye than the other. Table 1. dcr-1-enriched proteins detected in Drosophila oocytes DIGE can detect protein differences as low as Ϯ1.2-fold (24). by DIGE Master DIGE gels revealed a total of 94 difference proteins of Protein CG Fluorescence ratio* Relative MASCOT 1,003 detected proteins with changed abundance between wild- spot identiﬁer (dcr-1͞wild type) abundance† score‡ type and dcr-1 samples. Dye reversal of protein homogenates revealed the same spectrum of difference proteins. Two differ- 1 CG1782 3.6 2.6E-2 194 ence proteins (spots 51a and -b) located close to each other 2 CG5355 2.6 6.7E-2 209 changed reciprocally (Fig. 3, which is published as supporting 3 CG4264 3.7 1.9E-1 244 information on the PNAS web site), and MS analysis confirmed 4 CG12101 14.0 1.3E-2 82 these were related isoforms of the same gene product encoded 5 CG10602 4.2 1.2E-2 67 by CG1516. Of the remaining 92 difference proteins, 51 were 7 CG5384 2.8 1.0E-2 91 more abundant in wild-type samples (Fig. 3), and 41 difference 8 CG17654 34.0 1.3E-3 82 proteins were more abundant in dcr-1 mutant samples (Fig. 1 C 10 CG4916 7.0 1.0E-2 63 and D). We concentrated further analysis on the latter category, 11 CG2985 2.7 1.0 100 the dcr-1-enriched proteins, because these difference proteins 12 CG4027 4.3 1.4E-1 124 were repressed in the presence of Dcr-1. 13 — 3.3 9.5E-2 We quantified protein abundance by digital imaging of the Cy3 14 CG6084 5.6 3.8E-3 116 and Cy5 fluorescence of each relevant spot, using a charge- 15 CG4904 3.2 2.6E-2 153 coupled device camera that detects linear light intensity over 4 17 — 3.5 1.5E-2 orders of magnitude (20). The fluorescence ratios of the dcr-1- 19 — 3.3 9.0E-3 enriched proteins were compared with the normalized ratio, and 20 — 2.0 4.0E-1 all were found to be Ͼ2SD(Ϯ50%) different from the nor- 25 CG4463 1.7 2.3E-2 100 malized factor (Fig. 1E). Thus, the difference proteins exhibit 26 — 3.5 3.2E-3 significantly greater abundance in dcr-1 than wild-type samples 27 — 4.0 1.1E-2 (P Ͻ 0.05). The ratios of dcr-1 vs. wild-type fluorescence were 31 — 2.5 3.0E-3 expressed as a fold difference and are listed for each difference 32 CG7052 3.2 5.0E-3 69 protein (Table 1). The fold difference ranged from 1.6- to 33 CG7033 1.6 6.5E-2 160 69-fold; the average was 3.5-fold. The relative abundance of 34 — 2.3 2.5E-2 difference proteins varied over 3 orders of magnitude, as de- 35 — 2.4 1.2E-2 termined by the fluorescence intensity of wild-type proteins 36 CG4535 4.0 3.7E-2 90 (Table 1). 37 CG4422 3.3 4.1E-2 139 38 — 2.0 5.7E-2 Identification of Difference Proteins. Each of the dcr-1-enriched 39 — 5.4 1.6E-3 proteins was excised, subjected to in-gel tryptic digestion, and 40 — 6.0 1.6E-2 analyzed by MALDI-TOF MS. Twenty-two of the 41 proteins 41 — 2.9 3.5E-2 were identified by MS (Table 1). Many dcr-1-enriched proteins 42 — 3.2 2.1E-2 function in protein metabolism (Table 2), with two playing direct 43 CG3416 10.1 1.3E-3 104 roles in protein synthesis. stubarista encodes rp-S2, whereas 44 CG4634 69.0 5.0E-4 62 me31B encodes a DEAD-box ATPase regulator of translation 46 — 4.0 2.2E-2 (26, 27). One highly represented category functions in protein 47 — 3.2 1.9E-2 folding. Four protein chaperones were identified, and the 48 — 3.4 1.7E-2 FKBP59 proline isomerase, which is active in the embryo, was 49 — 3.8 2.2E-2 detected as well (28). The GDP dissociation inhibitor is a factor 50 CG17820 52.0 1.0E-3 83 important for intracellular protein transport (29). Another 68 CG14792 3.8 4.8E-2 83 highly represented category functions in protein turnover. Uba1 92 CG2979 2.9 6.7E-1 201 encodes an E1 ubiquitin-activating enzyme, whereas Pros35 and 93 — 2.2 3.1E-1 Mov34 encode subunits of the 26S proteasome (30). A protein M ϭ 3.5§ M ϭ 2.2E-2§ with putative ubiquitin-specific protease activity (31) and an *Sum of pixel values in spot from one channel over sum from other channel, M1-class metallopeptidase were also identified. In addition to corrected for background. those proteins that function in protein metabolism, a few pro- †Sum of pixel values in spot from wild-type sample relative to most abundant teins are enzymes involved in carbohydrate and lipid metabo- spot (11). lism. Enolase and aldehyde reductase are involved in glucose ‡A MASCOT score value Ͼ58 is considered signiﬁcant (P Ͻ 0.05) (38). metabolism, and Nurf-38 encodes a pyrophosphatase. Yp1 and §The median average for the 41 values listed. Yp2 are yolk-associated lipases that catabolize yolk lipids. Other difference proteins included actin, TepII [a ␣-2-macroglobulin protein (32)], and Fit, a protein of unknown function. clear whether the repression was a direct effect of miRNAs. If We also excised and analyzed 31 dcr-1-depleted proteins that it is direct, oocytes should contain mRNA transcripts encoding were more abundant in wild-type samples. MS identification of these proteins, because transcripts are the substrates for miRNA 21 of these proteins revealed that they have a variety of bio- binding. This eliminated Yp1 and Yp2 from consideration, chemical activities, with the majority functioning in carbohy- because these genes are transcribed in somatic cells of the drate metabolism and transport (Table 4, which is published as female, and their protein products are transported to the oocyte supporting information on the PNAS web site). The dcr-1- (33). To test the other genes, we measured levels of each depleted proteins are presumably regulated one or more steps transcript by quantitative RT-PCR, and we detected transcripts downstream of miRNA action, because their expression is for 19 genes in mature wild-type eggs (Fig. 2 and Table 2). No up-regulated by the silencing machinery. transcript was detected for Fit, which is expressed in the maternal fat body (34).

Comparison of Transcript and Protein Levels. The dcr-1-enriched If the 19 transcripts we detected are translationally repressed BIOCHEMISTRY proteins appear to be repressed by miRNAs. However, it was not by miRNAs, their abundance should not be greater in the dcr-1

Nakahara et al. PNAS ͉ August 23, 2005 ͉ vol. 102 ͉ no. 34 ͉ 12025 Downloaded by guest on September 29, 2021 Table 2. Functional and regulatory annotation of identiﬁed dcr-1-enriched proteins CG Transcript ratio† Protein͞transcript identiﬁer Gene* Function (dcr-1͞wild type) (dcr-1͞wild type)‡

CG4264 Hspc4 Chaperone 0.88 3.6 CG12101 Hsp60 Chaperone 0.59 24.1 CG4463 Hsp23 Chaperone 0.95 1.8 CG7033 — GroEL chaperone 0.63 2.6 CG4535 FKBP56 Prolyl cis-trans isomerase 0.75 5.4 CG4422 GDI Rab-GDP regulator 0.58 5.6 CG14792 stub rpS2 ribosomal subunit 0.19 31.7 CG4916 Me31B DEAD-box ATPase 0.97 8.6 CG1782 Uba1 Ubiquitin activating enzyme 0.54 6.6 CG5384 — Ubiquitin speciﬁc protease 0.82 3.4 CG4904 Pros35 Proteasome subunit 0.80 5.2 CG3416 Mov34 Proteasome subunit 0.08 125.0 CG5355 — Serine protease 2.10 1.2 CG10602 — M1 metalloprotease 0.84 5.1 CG7052 TepII Toll pathway protease inh. 0.20 15.6 CG6084 — Aldchyde reductase 0.40 10.8 CG17654 Enolase Pyruvate hydratase 0.88 38.4 CG4634 Nurf38 Inorganic pyrophosphatase 1.38 50.4 CG4027 Actin5C Cytoskeleton protein 0.66 5.6 CG2985 Yp1 Yolk protein — — CG2979 Yp2 Yolk protein — — CG17820 ﬁt Unknown — —

*FLYBASE entry (http:͞͞ﬂybase.bio.indiana.edu). †RT-PCR levels from mutant and wild-type RNA, normalized to rp49 as control. ‡Fluorescence ratio from Table 1 adjusted using the transcript ratio.

mutant than in wild-type. When we compared the transcript mutant. In summary, 18 transcripts appeared to be candidate levels in wild-type and dcr-1 individuals, 18 of the 19 transcripts substrates for translational inhibition by miRNAs. The strength were not significantly increased in the mutant (Fig. 2 and Table of inhibition, as measured indirectly as the ratio of protein to 2). One exception was the gene CG5355, which showed an transcript abundance, varied between 1.8- and 125-fold, with increased transcript level in the mutant that was quantitatively little ranking by functional class (Table 2). similar to its increase in protein abundance. This suggests that Dcr-1 primarily represses CG5355 through transcript production Detection of miRNA-Binding Sites. Transcripts that are directly or stability. For the other 18 transcripts, several were actually regulated by miRNAs should contain miRNA-binding sites in reduced in dcr-1 mutants, even though their protein products their 3Ј UTRs. Functional binding sites typically pair to cognate showed enhanced accumulation by DIGE. This might be due to miRNAs with extensive gaps and base mismatches (5). However, the enhanced translation in the mutant resulting in greater a six- to eight-base ‘‘seed’’ of contiguous pairing exists between mRNA turnover. Alternatively, reduced transcript abundance the 3Ј end of a binding site and 5Ј end of a miRNA (18). Another might result from disruption of other regulatory pathways in the feature of known functional binding sites is that they are

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.

12026 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500053102 Nakahara et al. Downloaded by guest on September 29, 2021 Table 3. Predicted miRNA-binding sites in 3؅ UTRs of Indeed, we found an average of 0.2 binding sites per gene in the identiﬁed genes depleted class (data not shown), in contrast to the 2.4 sites per Z score, gene in the enriched class. A second method we applied to ͞ Ј determine significance was a dual statistical test. We calculated Gene CG 3 UTR Predicted miRNA S score, binding ⌬ number (bases) sites (miR-) site number energy the probability that the MFOLD G value for each miRNA- binding interaction was greater than random average (Table 4). Hspc4 302 280, 275 3.1 2.1 This Z score was considered statistically significant (P Ͻ 0.05) if Hsp60 483 280, 1,* 5, 11, 6.1 5.3 it was Ͼ2.0, which indicates that it is at least 2-fold greater than 31a the standard deviation for background measurements. We also Hsp23 209 4, 305, 308 9.4 2.8 calculated the probability of finding the seed sequences in each CG7033 437 280(2),† 1, 305 11.6 8.4 3Ј UTR by chance. The resulting S score was considered FKBP56 130 280(2)‡ 1.9 2.5 significant (P Ͻ 0.05) if it was Ͼ2.0, again indicating that it is at GDI 166 280, 2b, 33, 287 8.3 10.5 least 2-fold greater than the standard deviation for randomly Stub 96 280 13.9 2.4 finding those seeds. Using these criteria, we conclude that most Me31B 127 280‡ 0.6 1.1 of the genes that we identified by proteomic analysis are pre- Uba1 481 280, 1, 5, 14 5.0 5.6 dicted to contain specific binding sites for miRNAs. CG5384 69 280‡ 0.9 3.6 Many of the binding sites identified in our analysis were also Pros35 107 12‡ 0.9 2.8 predicted from published computational analyses of the Dro- Mov34 254 280(2), 92b, 308 9.3 3.2 sophila melanogaster genome (13, 14) (Table 3). Despite this CG10602 129 280‡ 0.8 0.9 apparent concordance, we did not detect a few binding sites TepII 108 None predicted from those analyses. Moreover, several binding sites CG6084 269 280† 0.3 1.9 that our analysis identified were not found in the other analyses. Enolase 370 280, 8,* 308 6.2 2.2 These analyses considered only sites conserved in more than one Nurf38 137 4, 79 4.7 2.3 Drosophila species. Failure to identify sites might have occurred Actin5C 349 280, 1, 287, 288 9.9 9.9 because orthologous genes were not found, as happened with 35% of genes. Alternatively, sites in D. melanogaster genes might *miRNA-binding site predicted by published genome-wide computational searches (13, 14) that were not predicted by the present analysis. not be conserved in the other species. †Number in parentheses refers to number of binding sites found for the listed miRNA. Discussion ‡Indicates a miRNA-binding site predicted by the present analysis that was not At the completion of Drosophila oocyte maturation, the number predicted by published computational searches (13, 14). of genes negatively regulated by miRNAs appears to be limited. We detected 41 of 1,003 proteins that are down-regulated by miRNA production, which represents a maximum 4% of genes ⌬ predicted to bind with greater free energy ( G) to a cognate that might be directly inhibited. Although this value is close to miRNA than to an RNA of random sequence. Two other the genome-wide 7% level predicted by sequence comparisons qualities have been noted about functional miRNA-binding sites. (13), we cannot be certain that 4% represents a typical fraction First, binding sites are frequently repeated within a 3Ј UTR, and of genes regulated by miRNAs in a cell. First, this estimate binding sites for multiple miRNAs can be found within a UTR. reflects only the 1,000 most-abundant proteins at this stage of Second, conserved binding sites can be found in the 3Ј UTRs of development. Many less-abundant proteins are not detected by homologous genes of related species. DIGE, and so we do not know the fraction of those that are We developed a search algorithm to find potential miRNA- regulated. For example, dcr-1 mutant oocytes exhibit defective binding sites in RNA sequence. This algorithm searches for translation of Oskar and Gurken proteins, leading to a mild motifs based on the most recent discoveries about binding-site ventralization phenotype (data not shown). However, these two features. These are (i)a7͞8 base-pairing match or better proteins are too rare to have been detected by our proteomic between the 5Ј end of a miRNA and 3Ј end of the target site, with analysis. Second, we do not know whether the relative fraction no more than one permitted GU wobble pair; (ii)anMFOLD (35) of target genes varies during development. Possibly, fewer genes ⌬G value for each miRNA-binding interaction that is greater are regulated by miRNAs during oogenesis. It is known that than random average. We scanned the 3Ј UTR sequences of the RNAi is activated during oocyte maturation, and that the short 18 dcr-1 enriched-genes for putative miRNA-binding sites. We interfering RNA pathway depends upon translation of target searched only for sites recognized by a miRNA that is possibly mRNAs (23). It is possible that there are fewer miRNA targets expressed in mature oocytes or early embryos based on Northern in mature oocytes due to dependence on different translation analysis. control mechanisms. The results of the analysis are shown in Table 3. All but 1 of Why are these particular gene products repressed by miRNAs the 18 genes contains putative miRNA-binding sites in their 3Ј during oocyte maturation? In most animal species, translation UTRs. The average number of binding sites is 2.4 per gene, and serves as the main mechanism to regulate gene expression during the number of sites varies from one to four. The predicted list of oocyte maturation and early embryogenesis (36). Indeed, oocyte miRNAs is striking. miR-280 is predicted to interact with 14 of maturation and early embryogenesis proceed without transcrip- the 18 genes. This suggests that miR-280 is an important medi- tion of nuclear RNA, including rRNA. In parallel, no rps are ator of translation regulation at this stage of Drosophila devel- synthesized de novo, and consequently no new ribosomes are opment. Indeed, highly ranked target predictions for miR-280 produced. In Drosophila, rp-mRNA levels are constant through- include several genes that function in late oogenesis͞early out oogenesis and embryogenesis, but their translation drops as embryogenesis, including Toll, staufen, nudel, germ cell-less, oogenesis ends and embryogenesis begins (37, 38). Translation oskar, and par-6 (13, 14, 17). of rp-mRNAs then rises in conjunction with the onset of rRNA We determined the significance of these sites by two methods. transcription in the embryo. We have found that the accumu- First, we found that the presence of putative binding sites lation of rp-S2 protein is inhibited by miRNAs during oocyte correlated with genes that are down-regulated by Dcr-1. We maturation, suggesting that the translation block exerted on

scanned the dcr-1-depleted genes, reasoning that few of these Drosophila rp proteins is partially mediated by the miRNA BIOCHEMISTRY genes should contain miRNA-binding sites in their 3Ј UTRs. pathway. We do not know the status of other r-proteins, because

Nakahara et al. PNAS ͉ August 23, 2005 ͉ vol. 102 ͉ no. 34 ͉ 12027 Downloaded by guest on September 29, 2021 most are basic in charge and would not be detected in the 2D gels be replenished. Another possibility is that reduced proteolysis used in the DIGE analysis. links the rate of protein biogenesis to the rate of protein A lack of new ribosome production during oocyte maturation turnover, thereby maintaining steady-state protein levels. Fi- and early embryogenesis would also eliminate the need to nally, lowered protein turnover during this highly dynamic stage synthesize other factors involved in protein biogenesis, reflecting of development would allow for rapid and global accumulation a coordinated effort to balance various steps along the biogenesis of new proteins necessary for early embryogenesis. pathway. Our study indicates that the synthesis of several chaperones and other biogenic factors is specifically inhibited during We thank D. Marks and C. Sander for helpful input, members of the oocyte maturation, possibly for those reasons. Another cellular J.S.M. laboratory for help and advice, and E. Sontheimer and Y. S. Lee for greatly improving the manuscript. This work was supported by Grant process that appears to be attenuated by miRNAs at this GM07345 from the National Institutes of Health awarded to R.W.C. The developmental stage is protein turnover. Why is proteolysis MALDI-TOF MS in the Center for Molecular Analysis at Carnegie dampened? Possibly, reduced proteolysis allows preexisting ri- Mellon University is supported by National Science Foundation Grant bosomes to remain abundant during the period when they cannot CHE-9808188.

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