Small Peptides Switch the Transcriptional Activity of Shavenbaby During Drosophila Embryogenesis

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Science  16 Jul 2010:
Vol. 329, Issue 5989, pp. 336-339
DOI: 10.1126/science.1188158


A substantial proportion of eukaryotic transcripts are considered to be noncoding RNAs because they contain only short open reading frames (sORFs). Recent findings suggest, however, that some sORFs encode small bioactive peptides. Here, we show that peptides of 11 to 32 amino acids encoded by the polished rice (pri) sORF gene control epidermal differentiation in Drosophila by modifying the transcription factor Shavenbaby (Svb). Pri peptides trigger the amino-terminal truncation of the Svb protein, which converts Svb from a repressor to an activator. Our results demonstrate that during Drosophila embryogenesis, Pri sORF peptides provide a strict temporal control to the transcriptional program of epidermal morphogenesis.

Studies of eukaryotic genomes have revealed that a large proportion of genomic DNA produces atypical long transcripts, the functions of which are controversial (14). These transcripts contain only short open reading frames (sORFs, <100 codons) and thus are generally considered to be non-protein-coding RNAs (ncRNAs). However, there is growing evidence that the sORFs present in some ncRNAs are actually translated into small peptides, the abundance of which is probably greatly underestimated (57). Whereas sORF-encoded peptides may represent an overlooked repertoire of bioactive molecules (8), their functions and the mechanisms by which they operate are largely unknown.

We and others recently identified an evolutionarily conserved sORF gene, referred to as polished rice (pri) or tarsal-less (tal) in Drosophila, and mille-pattes (mlpt) in Tribolium (911). pri mRNA is a polycistronic transcript that encodes four similar peptides, 11 to 32 amino acids in length, that play a redundant role in Drosophila embryogenesis (9, 10). Embryos that lack pri display prominent defects, including the absence of trichomes and aberrant tracheal architecture (9, 10). Reduced pri activity in imaginal development results in abnormal leg morphogenesis (10, 12). Similarly, mlpt knockdown in Tribolium leads to appendage defects and the transformation of segmental identity (11).

To gain insight into the molecular function of Pri peptides, we focused on their role in trichome formation during Drosophila embryogenesis. Epidermis differentiation results in a pattern of smooth cells and cells that form apical extensions, called trichomes (ventral denticles and dorsal hairs) (Fig. 1A) (13). Modifications of the trichome pattern that have been examined in insects (resulting from laboratory-induced mutations or evolutionary diversification) are so far all attributable to changes in expression of shavenbaby (svb) (1416). Indeed, svb encodes a transcription factor that directly regulates the expression of target effectors, which are collectively responsible for trichome formation (17, 18). Although the absence of pri results in trichome loss, the expression of svb is not altered in pri mutants (9). Reciprocally, pri is expressed normally in svb mutants (9), showing that svb and pri act in parallel in trichome formation (fig. S1). Expression of Svb target genes, such as miniature and shavenoid (17), is lost in pri mutants, whereas the expression of other epidermal genes is unaffected (Fig. 1B and S2). The activity of isolated Svb-responsive enhancers was also strongly reduced in pri mutants (fig. S3). Therefore, pri is specifically required for the transcription of Svb downstream targets in trichome cells.

Fig. 1

pri is required for the expression of Svb target genes. (A) Embryonic cuticle specimens, showing the two types of trichomes, ventral denticles (arrowheads and middle close-up) and dorsal hairs (bracket and right close-up). (B) Two Svb downstream targets, miniature (m) and shavenoid (sha) are expressed in trichome cells at stages 15 and 16 in wild type, but not in pri and svb mutants. As a control of pri specificity for Svb function, the epidermal expression of CG16885 (independent of svb) was not affected in pri mutants. Anterior is to the left and dorsal is to the top, except for the close-ups in (A). Scale bar, 10 μm.

How can Pri peptides regulate the expression of Svb target genes without affecting svb expression? The svb locus encodes three overlapping protein isoforms: Svb and the germline-specific proteins OvoA and OvoB (Fig. 2A) (19, 20). Ovo/Svb proteins all share the same DNA-recognition and transcriptional-activation domains but differ in their N-termini (Fig. 2A). The shortest isoform, OvoB, is a transcriptional activator and induces trichomes when artificially expressed in the epidermis (Fig. 2F) (19). OvoA contains an extended N-terminal region, which switches its function toward active transcriptional repression and thus dominantly inhibits trichome formation (Fig. 2C) (19). Svb contains a further N-terminal extension, compared to OvoA, and promotes the formation of ectopic trichomes like OvoB (Fig. 2D) (20). To evaluate the specificity of Pri/Svb interaction, we examined the influence of pri on the different Ovo/Svb isoforms with respect to trichome formation. In wild-type embryos, seven rows of ventral cells per segment express svb and form trichomes (Fig. 2B) (13, 15). Upon its ectopic expression in smooth cells, Svb [or Svb:green fluorescent protein (GFP)] induced supernumerary trichomes in control embryos (Fig. 2D) but not in pri mutants (Fig. 2E). In contrast, OvoB (or OvoB:GFP) was insensitive to pri, with ectopic trichomes forming in both control and pri mutant embryos (Fig. 2, F and G). In the latter case, we observed only OvoB-induced ectopic trichomes and no Svb-dependent endogenous trichomes (Fig. 2G). These results show that whereas pri has no effect on the shorter OvoB isoform, pri peptides specifically control the ability of Svb to induce trichomes.

Fig. 2

The ability of Svb to induce trichomes depends on pri. (A) Scheme of the ovo/svb locus and protein isoforms. Coding (CDS) and untranslated regions (UTR) of mRNAs are represented by blue and white boxes, respectively. The Svb-specific protein region is in turquoise; the repression, activation, and DNA-binding domains are in red, green, and gray, respectively. (B to G) Micrographs of ventral trichomes (A4 segment) and illustrations of epidermal cells expressing svb (purple), ovoA (red), and ovoB (green) in embryos of different backgrounds. pri mRNA is widely expressed in epidermis and reinforced in trichome cells (fig. S1F). (B) Wild-type embryo. (C) Embryo expressing UAS-ovoA:GFP under the control of ptc-GAL4. (D) and (E) Embryos expressing UAS-svb:GFP under the control of wg-Gal4 in the presence (D) or absence (E) of pri. (F) and (G) Embryos expressing UAS-ovoB:GFP under the control of wg-Gal4 in the presence (F) or absence (G) of pri. Red brackets indicate ectopic trichomes. Scale bar, 10 μm.

We next examined whether Pri peptides affect the synthesis or trafficking of Ovo/Svb proteins. Using transgenic C-terminal GFP-fusions (proven functional as described above), we observed that pri does not influence the production of Ovo/Svb proteins or their import to the nucleus (Fig. 3 and fig. S4). However, we noticed different patterns of their intranuclear distribution. Regardless of pri activity, throughout embryogenesis OvoA accumulated in discrete foci (Fig. 3A), and OvoB was distributed diffusely in the nucleoplasm (Fig. 3B). During stages 11 and 12, before pri is expressed in the epidermis (fig. S1), Svb formed intranuclear foci, like OvoA (Fig. 3C). At the onset of pri epidermal expression (stage 13 onwards) (fig. S1), the nuclear distribution of Svb became diffuse (Fig. 3C). Therefore, Svb distribution changes from a pattern similar to the OvoA repressor to that of the OvoB activator, and the timing of this conversion correlates with the expression of pri. This redistribution of Svb was abolished in pri mutants, in which Svb remained in nuclear foci throughout embryogenesis (Fig. 3C). Thus, pri participates in the conversion of nuclear distribution of Svb from punctate to diffuse.

Fig. 3

pri regulates the subnuclear localization of Svb in living embryos. Distribution of (A) OvoA:GFP, (B) OvoB:GFP, and (C) Svb:GFP driven by wg-GAL4 in control (pri +/−) or pri mutant embryos at stages 11 and 12 and stages 13 to 16. In all cases, the GFP signal was restricted to nuclei (fig. S4). Although the distribution of OvoA (focal) and OvoB (diffuse) was insensitive to pri, Svb switched from foci to diffuse in a pri-dependent manner. Scale bar, 10 μm.

The nonpunctuated, diffuse nuclear distribution of Svb in epidermal cells correlates with its ability to induce trichomes, suggesting that Svb redistribution coincides with active transcription of its targets. We explored this hypothesis using assays in Drosophila Schneider cells (S2 cells), which are of embryonic origin. Similarly to observations in embryos, the nuclear pattern of Svb was converted from punctate to diffuse in a pri-dependent manner in S2 cells (Fig. 4A). We quantified the transcriptional activity of Svb/Ovo using the Enh-m enhancer, which is directly activated by Svb in vivo (17) (fig. S3). OvoB strongly stimulated the transcription driven by Enh-m, and OvoA repressed transcription, both with or without pri (Fig. 4B). In contrast, Svb behaved like OvoA in the absence of pri, but similar to OvoB, activated Enh-m in the presence of Pri peptides (Fig. 4B). Inactivation of the Svb-binding site of Enh-m (17) suppressed this activation (fig. S5), indicating that pri is required for the direct activation of Enh-m by Svb. These results demonstrate that Pri peptides switch the transcriptional activity of Svb from that of a repressor accumulated in nuclear foci to a nucleoplasmic activator.

Fig. 4

Pri converts the Svb protein from a transcriptional repressor to an activator by N-terminal truncation. (A) Subnuclear localization of Svb:GFP in S2 cells when pri is co-expressed. 1-4FS, a full-length pri mRNA with frame-shift mutations in ORF1-4 (9), was used as control. Cells were stained with antibody to GFP (green) and anti-Svb1s (red). Nuclei are in blue. The rightmost panel is a three-dimensional representation of Svb nuclear distribution. (B) Transcriptional activity of OvoA, OvoB, and Svb in S2 cells. Luciferase activity was used as a reporter for the transcriptional activity of the Enh-m enhancer. Error bars represent SE, and significance against GFP/1-4FS–transfected cells was evaluated with t tests (*P < 0.05, **P < 0.01, #P > 0.05). (C) Distribution of Svb:PA-GFP (green) in S2 cells, before photoactivation (t0), after photoactivation (t0’), and after the induction of pri expression (t18h). Without pri induction, Svb:PA-GFP was retained in foci (–pri, t18h). Red is Moesin:red fluorescent protein (RFP) used as a transfection control. (D) Western blots analysis of S2 cells expressing OvoA:GFP, OvoB:GFP, and Svb:GFP. Protein extracts were immunoprecipitated with antibody to GFP and probed with antibody to Ovo or anti-Svb1s. (E) Schematic representation of predicted form of truncated Svb. The N terminus of truncated Svb matches the sequence AAGHGR, which is located 56 amino acids upstream of the OvoB-initiating methionine (asterisk). Red arrows indicate the regions used to generate antibodies. (F) Ventral views of wild-type and pri mutant embryos stained with anti-Svb1s (red), and antibody to Miniature (green) that underlies nascent trichomes. Nuclei are in blue. Scale bar, 10 μm.

To explore the mechanisms by which Pri peptides trigger this switch in Svb intranuclear distribution, we examined whether Pri requires de novo synthesis of the Svb protein. Using a photoactivatable-GFP (PA-GFP), we observed that photoactivated Svb:PA-GFP switched from foci to diffuse distribution after the induction of pri expression (Fig. 4C). Therefore, the same Svb molecules are relocated within the nucleus, suggesting that the action of Pri peptides relies on posttranslational modifications of Svb. Accordingly, Western blot analysis showed that whereas the size of OvoA and OvoB proteins (including that of their minor species) were not affected by pri, Svb exhibited a differential electrophoretic mobility in a pri-dependent manner (Fig. 4D). In the absence of pri, Svb appeared slightly larger than OvoA, as deduced from the cDNA sequences (Fig. 2A). Upon pri expression, the Svb protein displayed a faster mobility, corresponding to a truncation of approximately 50 kD, without apparent modification in the size of svb mRNA (fig. S6). An antibody raised against the N-terminal Svb-specific region (anti-Svb1s) recognized only the larger Svb protein but not the truncated product formed upon pri expression. This truncated Svb protein was detected by antibodies to Ovo and GFP (Fig. 4, A and D, and fig. S7A), showing that it lacks the N-terminal region but retains an intact C terminus. To further characterize Svb truncation, we purified the truncated Svb protein and micro-sequenced its N-terminal end (fig. S8A). The N terminus of truncated Svb matches the sequence AAGHGR, which is located 56 amino acids upstream of the OvoB-initiating methionine and within a protein region that shows strong evolutionary conservation in insects (Fig. 4E and fig. S8B). The corresponding DNA sequence displays synonymous nucleotide substitutions across species and lacks canonical or alternative initiation codons (fig. S8B), further supporting the view that Svb truncation results from a posttranslational cleavage. Hence, the pri-induced truncated form of Svb contains the DNA-binding and activation domains but not the repression domain, explaining why it acts as a transcriptional activator.

Consistent with this idea, we observed a pri-dependent truncation of the endogenous Svb protein during embryogenesis. In wild-type embryos, anti-Svb1s detected a transient nuclear signal in trichome cells, at stages 11 and 12, that disappeared at later stages (Fig. 4F and fig. S7B). The loss of the Svb N-terminal region coincided with the onset of pri expression in the epidermis (fig. S1). Indeed, pri is required for Svb truncation in vivo—as revealed by the persistence of anti-Svb1s signal in pri mutants—throughout embryogenesis (Fig. 4F). We conclude that Pri peptides convert Svb from a transcriptional repressor to an activator via the truncation of its N-terminal region.

This study demonstrates that 11– to 32–amino acid peptides encoded by sORFs orchestrate epidermal differentiation through the control of Svb transcriptional activity. At stages 11 and 12, svb is already expressed and restricted to presumptive trichome cells, in which the full-length Svb repressor probably prevents the premature expression of cellular effectors. At stages 13 and 14, the expression of pri in epidermal cells then triggers N-terminal truncation of the Svb protein, probably through a proteolytic release of the repressor domain, causing a rapid conversion of Svb function toward activation. Thus, although svb expression defines the spatial pattern of trichomes, the action of Pri peptides defines the temporality of trichome formation.

Besides the mechanisms of epidermal differentiation, our studies suggest broader functions for Pri peptides. Although pri is also required for tracheal morphogenesis (9), we observed normal trachea in svb mutant embryos (fig. S9), indicating that Pri peptides probably regulate additional developmental factors. Recent large-scale analyses indicate that thousands of unexplored transcripts are also probably encoding polypeptides of less than 100 amino acids in mice and humans (1, 21, 7). Future functional analyses should elucidate how small peptides encoded by transcripts improperly termed ncRNAs contribute to various biological processes including development and differentiation.

Supporting Online Material

Materials and Methods

Figs. S1 to S9


References and Notes

  1. We thank the Kyoto Drosophila Genetic Resource Center (DGRC); Bloomington Drosophila Stock Center for fly strains; the Indiana DGRC; G. Patterson, J. Lippincott-Schwartz, and C. Hill for plasmids; S. Takada and T. Okubo (NIBB) for technical advice; Y. Latapie and B. Ronsin (CBD) and J. D’Alayer (Institut Pasteur) for excellent technical assistance; and members of the Kobayashi laboratory for helpful comments and discussion. This work was supported by a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science; the JST PRESTO program; the Ministry of Education, Culture, Sports, Science and Technology KAKENHI (20370091); Agence Nationale de la Recherche (Programme Blanc “Netoshape”); Fondation pour la Recherche Médicale (Equipe 2005); and Association pour la Recherche sur le Cancer (1111).
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