Pri sORF peptides induce selective proteasome-mediated protein processing.

A wide variety of RNAs encode small open-reading-frame (smORF/sORF) peptides, but their functions are largely unknown. Here, we show that Drosophila polished-rice (pri) sORF peptides trigger proteasome-mediated protein processing, converting the Shavenbaby (Svb) transcription repressor into a shorter activator. A genome-wide RNA interference screen identifies an E2-E3 ubiquitin-conjugating complex, UbcD6-Ubr3, which targets Svb to the proteasome in a pri-dependent manner. Upon interaction with Ubr3, Pri peptides promote the binding of Ubr3 to Svb. Ubr3 can then ubiquitinate the Svb N terminus, which is degraded by the proteasome. The C-terminal domains protect Svb from complete degradation and ensure appropriate processing. Our data show that Pri peptides control selectivity of Ubr3 binding, which suggests that the family of sORF peptides may contain an extended repertoire of protein regulators.

Small peptides switch the transcriptional activity of Shavenbaby during Drosophila embryogenesis.

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.

Effects of mild heat shocks at young age on aging and longevity in Drosophila melanogaster.

Young adult flies were submitted to heat shocks (37 degrees C) of various durations (5, 10, 20, 40 or 60 min daily) for 1, 2 or 3 weeks. A slight longevity increase, in both sexes, was only observed with the lowest heat shock. Longer shocks had neutral or negative effects. Flies submitted to the procedure providing a longevity increase did not show a delayed behavioral aging but survived longer at 37 degrees C than control flies. This higher thermotolerance was not associated with an increased hsp70 induction. The results are discussed in connection with hormesis and previous results showing that hypergravity, an other mild stress, increases longevity and delays behavioral aging: different mild stresses may have contrasting effects on aging and longevity.

OVO transcription factors function antagonistically in the Drosophila female germline.

OVO controls germline and epidermis differentiation in flies and mice. In the Drosophila germline, alternative OVO-B and OVO-A isoforms have a common DNA-binding domain, but different N-termini. We show that these isoforms are transcription factors with opposite regulatory activities. Using yeast one-hybrid assays, we identified a strong activation domain within a common region and a counteracting repression domain within the OVO-A-specific region. In flies, OVO-B positively regulated the ovarian tumor promoter, while OVO-A was a negative regulator of the ovarian tumor and ovo promoters. OVO-B isoforms supplied ovo(+) function in the female germline and epidermis, while OVO-A isoforms had dominant-negative activity in both tissues. Moreover, elevated expression of OVO-A resulted in maternal-effect lethality while the absence of OVO-A resulted in maternal-effect sterility. Our data indicate that tight regulation of antagonistic OVO-B and OVO-A isoforms is critical for germline formation and differentiation.

ovo/svb integrates Wingless and DER pathways to control epidermis differentiation.

In Drosophila, as in mammals, epidermal differentiation is controlled by signalling cascades that include Wnt proteins and the ovo/shavenbaby (svb) family of zinc-finger transcription factors. Ovo/svb is a complex gene with two genetic functions corresponding to separate control regions: ovo is required for female germline development and svb for epidermal morphogenesis. In the Drosophila embryo, the ventral epidermis consists of the segmental alternance of two major cell types that produce either naked cuticle or cytoplasmic extrusions known as denticles. Wingless signalling specifies smooth cells that produce naked cuticle, whereas the activation of the Drosophila epidermal growth factor (EGF) receptor (DER) leads to the production of denticles. Here we show that expression of the ovo/svb gene controls the choice between these cell fates. We find that svb is a key selector gene that, cell autonomously, directs cytoskeletal modifications producing the denticle. The DER pathway promotes denticle formation by activating svb expression. Conversely, Wingless promotes the smooth cell fate through the transcriptional repression of svb by the bipartite nuclear factor Armadillo/dTcf. Our data indicate that transcriptional regulation of svb integrates inputs from the Wingless and DER pathways and controls epidermal differentiation.

HSP70 induction may explain the long-lasting resistance to heat of Drosophila melanogaster having lived in hypergravity.

In this study, we showed that in flies kept for 2 weeks at 1 (terrestrial gravity), 3 or 5 x g (hypergravity, HG) before transfer to 1 x g, resistance to heat remained higher in HG flies for several weeks after the transfer. The measurement of heat shock protein 70 (hsp70) indicated no induction of the protein in HG, but the study revealed that flies living in HG expressed more hsp70 only after being submitted to severe stress. The higher induction of hsp70 may explain the higher thermotolerance of these HG-treated young flies. Finally, an unknown protein was observed only in females. This protein may belong to a class of higher molecular weight hsp (hsp110), which have not previously been observed in Drosophila.

Transcriptional control of Drosophila bicoid by Serendipity delta: cooperative binding sites, promoter context, and co-evolution.

Concentration of maternal BICOID (BCD) establishes the anterior pattern in the Drosophila embryo. Successive deletions in the bcd promoter allowed us to localize an enhancer sequence in the 5'-UTR and a down-regulating element downstream of the ATG initiator codon, and identify a 49 bp region sufficient to drive transcription of a reporter gene specifically in nurse cells. This fragment contains two binding sites for the Serendipity (Sry) d zinc finger activator, that mediate its cooperative binding. Both sites (sdbs) are essential for bcd expression. Further analysis showed that the bcd promoter configuration is decisive for Sry d activating function. Replacement of sdbs by binding sites for Sry b, the Sry d paralog, restores bcd transcription in sry d mutant ovaries, demonstrating that the functional divergence between these two proteins during evolution was mainly driven by changes in their DNA-specific recognition properties, resulting in the control of separate developmental pathways.

Two types of zinc fingers are required for dimerization of the serendipity delta transcriptional activator.

The serendipity (sry) delta zinc finger protein controls bicoid gene expression during Drosophila melanogaster oogenesis. In addition, sry delta mutants display various zygotic phenotypes, ranging from abnormal embryogenesis to sex-biased adult lethality. We report here that sry delta is a sequence-specific transcriptional activator. A single sry delta consensus binding site (SDCS), in either orientation, is sufficient to promote transcription activation in cell culture, and multiple SDCSs mediate a strong synergistic activation, reflecting the cooperativity of sry delta binding to DNA. Further, several lines of evidence strongly suggest that sry delta binds to DNA as a dimer. While each of three point mutations located in the third zinc finger of sry delta drastically reduces its DNA binding affinity, a fourth mutation, located in the N-terminal region of the protein, specifically affects the cooperativity of DNA binding. This mutation reveals the functional importance of a putative Cys2/Cys2 zinc finger motif of a novel type, located outside the DNA binding domain. A systematic deletion analysis shows that interaction between this proposed Cys2/Cys2 motif and a classical Cys2/His2 zinc finger mediates homodimerization, which is required for DNA binding cooperativity.

ovo, a Drosophila gene required for ovarian development, is specifically expressed in the germline and shares most of its coding sequences with shavenbaby, a gene involved in embryo patterning.

Genetic analyses of Drosophila oogenesis have revealed the central role of ovo, a gene required for differentiation of the female germline. A number of recessive ovo mutations also affect the shavenbaby (svb) function required for late embryo patterning, suggesting a tight structural link between ovo and svb. By using various genomic probes for in situ hybridization to wild type and mutant embryos, we show that ovo indeed shares most of its coding sequences with svb. svb expression is detected early in the presumptive head region and later in each segment. It requires control elements located upstream of the ovo genomic region. ovo expresses abundant maternal RNAs which are uniformly distributed in early cleavage embryos. A fraction that lacks an alternative ovo-specific protein coding region (ORF 2b) is detected in pole cells. Expression of an ovo-specific lacZ reporter gene (ovoB) shows that ovo encodes a nuclear protein present in the germline of both sexes. Zygotic ovoB expression is first detected in embryos at around stage 17 and persists up to the adult stage. Our data show that the germline specific expression of ovo in females correlates with its function in oogenesis. This expression, however, is also observed in males in which ovo is not required.

Direct control of transcription of the Drosophila morphogen bicoid by the serendipity delta zinc finger protein, as revealed by in vivo analysis of a finger swap.

Determination of the anterior structures of the Drosophila embryo is under control of the maternal gene product Bicoid (bcd), which specifies distinct domains of embryonic gene expression in a concentration-dependent manner. We show here that bcd transcription is controlled by serendipity delta (sry delta), a zygotic-lethal zinc finger protein gene. This sry delta germ-line function was revealed by transgenic expression of a modified Sry delta protein, Sry DB56, carrying a two-finger swap. Although it almost fully rescues sry delta lethality, Sry DB56 does not substitute for the wild-type protein in activating bcd transcription. Two overlapping sites binding the Sry delta protein were identified in the bcd promoter region, a few base pairs upstream of the putative TATA box. Mutating one site impairs bcd transcription in vivo, indicating that Sry delta acts directly upstream of bcd. The specific requirement of sry delta for bcd transcription in the female germ line constitutes an unexpected link between a zygotic gene with pleiotropic functions and the establishment of coordinates of the Drosophila egg. It highlights the fundamental role of ubiquitous transcription factors in bringing about a specific developmental program.

Zinc fingers and other domains cooperate in binding of Drosophila sry beta and delta proteins at specific chromosomal sites.

The closely related Drosophila serendipity (sry) beta and delta zinc finger proteins display consensus in vitro DNA recognition sequences differing by 4 of 13 nucleotide positions and bind in vivo to distinct sets of sites on polytene chromosomes. We compared the pattern of in vivo chromosomal binding of deleted forms of the sry delta protein fused to beta-galactosidase and expressed in Drosophila transgenic lines. Results show that the carboxy-terminal DNA-binding finger domain is required and sufficient for binding at specific chromosomal sites but that this binding does not nearly reproduce the wild-type pattern. An NH2-terminal domain of the sry delta protein is essential to its specificity of in vivo interaction with chromatin. In vitro and in vivo experiments using reciprocal finger swap between the sry beta and delta proteins suggest that the in vivo specificity is dependent on selective protein-protein contacts at defined chromosomal sites, in addition to DNA specific recognition.

Genomic targets of the serendipity beta and delta zinc finger proteins and their respective DNA recognition sites.

The closely related Drosophila serendipity (sry) beta (beta) and delta (delta) Cys2-His2 zinc finger proteins show partly overlapping in vitro DNA binding specificities and distinct patterns of binding sites on polytene chromosomes. Using a newly developed procedure, we identified genomic DNA targets for these two proteins. Both the sry beta and delta proteins protect an 18-22 base region from DNase I digestion within each analysed genomic binding site, that includes a 13 bp consensus sequence. The consensus recognition sites sry beta 5'-YCAGAGATGCGCA-3' and sry delta 5'-YTAGAGATGGRAA-3' thus differ by nucleotides at four out of 13 positions. They are determinant for specific binding of the sry beta and delta proteins, respectively, produced in Escherichia coli or present in Drosophila embryos. We further show that sry beta is the major (if not exclusive) Drosophila nuclear protein that specifically binds the 5'-CAGAGTGCGC-3' sequence. The identified sry beta genomic targets are all contained within single-copy DNA in euchromatic regions of the genome. Two out of the five characterized in detail map at cytological positions coincident with binding sites of the native sry beta protein on polytene chromosomes.

The closely related Drosophila sry beta and sry delta zinc finger proteins show differential embryonic expression and distinct patterns of binding sites on polytene chromosomes.

Serendipity (sry) beta (beta) and delta (delta) are two finger protein genes resulting from a duplication event. Comparison of their respective protein products shows interspersed blocks of conserved and divergent amino-acid sequences. The most extensively conserved region corresponds to the predicted DNA-binding domain which includes 6 contiguous fingers; no significant sequence conservation is found upstream and downstream of the protein-coding region. We have analysed the evolutionary divergence of the sry beta and delta proteins on two separate levels, their embryonic pattern of expression and their DNA-binding properties in vitro and in vivo. By using specific antibodies and transformant lines containing beta-galactosidase fusion genes, we show that the sry beta and sry delta proteins are maternally inherited and present in embryonic nuclei at the onset of zygotic transcription, suggesting that they are transcription factors involved in this process. Zygotic synthesis of the sry beta protein starts during nuclear division cycles 12-13, prior to cellularisation of the blastoderm, while the zygotic sry delta protein is not detectable before germ band extension (stage 10 embryos). Contrary to sry delta, the zygotic sry beta protein constitutes only a minor fraction of the total embryonic protein. The sry beta and delta proteins made in E. coli bind to DNA, with partly overlapping specificities. Their in vivo patterns of binding to DNA, visualised by immunostaining polytene chromosomes, differ both in the number and position of their binding sites. Thus changes in expression pattern and DNA-binding specificity have contributed to the evolution of the sry beta and delta genes.

Serendipity delta, a Drosophila zinc finger protein present in embryonic nuclei at the onset of zygotic gene transcription.

Serendipity delta (sry delta) is a member of a set of Drosophila zinc finger protein genes showing maximal transcription during oogenesis. By using transformant lines, we monitored the zygotic expression of the sry delta gene and characterized some biochemical properties of a sry delta/beta-galactosidase fusion protein-containing fingers. Further analysis made use of anti-sry delta specific antibodies. During oogenesis, while sry delta mRNAs transcribed by nurse cells are transferred to the oocyte starting in stage 10, translation into protein occurs in the ooplasm starting in stage 12. The maternally inherited protein concentrates in embryonic nuclei during early cleavages, prior to the onset of zygotic transcription. At the blastoderm stage, the sry delta protein is localized in all somatic nuclei. Later in embryogenesis and up to the adult stage, the zygotic protein is present in nuclei of transcriptionally active cells (both somatic and germ line). These data are consistent with the sry delta protein being a transcription factor, with a role in zygotic activation of general cellular functions.

cis-regulatory elements of the Drosophila blastoderm-specific serendipity alpha gene: ectopic activation in the embryonic PNS promoted by the deletion of an upstream region.

The Drosophila serendipity alpha gene (sry alpha) is specifically expressed at the blastoderm stage in all somatic cells. By deletion analysis of sry alpha-lacZ fusion genes, the sry alpha cis-acting regulatory elements have been restricted to the [-311, +130] 5'-region of the gene and separated in two domains. The [-118, +130] domain is sufficient for transcriptional activation at the blastoderm stage. The [-311, -118] domain is required for a full level of expression. Deletion of this upstream domain leads to a secondary pattern of lacZ expression in precursor cells of the peripheral nervous system (PNS). The sry alpha gene is not itself secondarily expressed in the PNS, as shown by in situ hybridization. The patterns of expression of the different sry alpha-lacZ fusion genes suggest a combinatorial mode of regulation of sry alpha expression at blastoderm.

Finger proteins and DNA-specific recognition: distinct patterns of conserved amino acids suggest different evolutionary modes.

Finger proteins, the first example of which was Xenopus TFIIIA, share Zn2+ finger-like folded domains capable of binding to nucleic acids. A large number of this type of protein have been characterised from diverse organisms, indicating a wide evolutionary spread of the DNA-binding fingers. At least two classes of finger proteins may be distinguished. Class I proteins contain variable numbers of the tandemly repeating TFIIIA-like finger motif, (Y/F-X-C-X2-4-C-X3-F-X5-L-X2-H-X3-H). Class II finger proteins display a single (C-X2-C-X13-C-X2-C) motif and a facultative second putative finger. The relation between the structure of finger proteins and their recognised DNA sequences is discussed.