The TopoVIB-Like protein family is required for meiotic DNA double-strand break formation.

Meiotic recombination is induced by the formation of DNA double-strand breaks (DSBs) catalyzed by SPO11, the ortholog of subunit A of TopoVI DNA topoisomerase (TopoVIA). TopoVI activity requires the interaction between A and B subunits. We identified a conserved family of plant and animal proteins [the TOPOVIB-Like (TOPOVIBL) family] that share strong structural similarity to the TopoVIB subunit of TopoVI DNA topoisomerase. We further characterize the meiotic recombination proteins Rec102 (Saccharomyces cerevisiae), Rec6 (Schizosaccharomyces pombe), and MEI-P22 (Drosophila melanogaster) as homologs to the transducer domain of TopoVIB. We demonstrate that the mouse TOPOVIBL protein interacts and forms a complex with SPO11 and is required for meiotic DSB formation. We conclude that meiotic DSBs are catalyzed by a complex involving SPO11 and TOPOVIBL.

Dominant modifiers of the polyhomeotic extra-sex-combs phenotype induced by marked P element insertional mutagenesis in Drosophila.

Members of the Polycomb group (Pc-G) and trithorax group (trx-G) of genes, as well as the enhancers of trx-G and Pc-G (ETP), function together to maintain segment identity during Drosophila development. In order to obtain new marked P mutations in these genes, we screened for dominant modifiers of the extra-sex-combs phenotype displayed by males mutant for the polyhomeotic (ph) gene, a member of the Pc-G group. Five P(lacW) insertions in four different genes were found to stably suppress ph: two are allelic to trithorax, one is the first allele specific to the Minute(2)21C gene, and the remaining two define new trx-G genes, toutatis (tou) in 48A and taranis (tara) in 89B10-13. tou is predicted to encode a 3109 amino acid sequence protein (TOU), which contains a TAM DNA-binding domain, a WAKZ motif, two PHD zinc fingers and a C-terminal bromodomain, and as such is likely to be involved in regulation of chromatin structure as a subunit of a novel chromatin remodelling complex. In a previous study, we found that insertion of a P(ph) transposable element containing ph regulatory sequences creates a high frequency of mutations modifying ph homeotic phenotypes. One such insertion enhanced the ph phenotype and we show that it is a new allele of UbcD1/eff, a gene encoding a ubiquitin-conjugating enzyme that is involved in telomere association and potentially in chromatin remodelling.

Distinctive features of Drosophila alternative splicing factor RS domain: implication for specific phosphorylation, shuttling, and splicing activation.

The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA precursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for both localization and splicing activity of hASF. While this difference has no effect on dASF localization, it impedes its capacity to shuttle between the nucleus and cytoplasm and abolishes its phosphorylation by SR protein kinase 1 (SRPK1). dASF also has an altered splicing activity. While being competent for the regulation of 5' alternative splice site choice and activation of specific splicing enhancers, dASF fails to complement S100-cytoplasmic splicing-deficient extracts. Moreover, targeted overexpression of dASF in transgenic flies leads to higher deleterious developmental defects than hASF overexpression, supporting the notion that the distinctive structural features at the RS domain between the two proteins are likely to be functionally relevant in vivo.

Drosophila homologs of transcriptional mediator complex subunits are required for adult cell and segment identity specification.

The origins of specificity in gene expression are a central concern in understanding developmental control. Mediator protein complexes regulate transcriptional initiation, acting as modular adaptors linking specific transcription factors to core RNA polymerase II. Here, we identified the Drosophila homologs of 23 human mediator genes and mutations of two, dTRAP240 and of dTRAP80 (the putative fly homolog of yeast SRB4). Clonal analysis indicates a general role for dTRAP80 necessary for cell viability. The dTRAP240 gene is also essential, but cells lacking its function are viable and proliferate normally. Clones reveal localized developmental activities including a sex comb cell identity function. This contrasts with the ubiquitous nuclear accumulation of dTRAP240 protein in imaginal discs. Synergistic genetic interactions support shared developmental cell and segment identity functions of dTRAP240 and dTRAP80, potentially within a common complex. Further, they identify the homeotic Sex combs reduced product, required for the same cell/tissue identities, as a functional partner of these mediator proteins.

The Drosophila poly(A)-binding protein II is ubiquitous throughout Drosophila development and has the same function in mRNA polyadenylation as its bovine homolog in vitro.

The poly(A)-binding protein II (PABP2) is one of the polyadenylation factors required for proper 3'-end formation of mammalian mRNAs. We have cloned Pabp2, the gene encoding the Drosophila homolog of mammalian PABP2, by using a molecular screen to identify new Drosophila proteins with RNP-type RNA-binding domains. Sequence comparison of PABP2 from Drosophila and mammals indicates that the most conserved domains are the RNA-binding domain and a coiled-coil like domain which could be involved in protein-protein interactions. Pabp2 produces four mRNAs which result from utilization of alternative poly(A) sites and encode the same protein. Using an antibody raised against Drosophila PABP2, we show that the protein accumulates in nuclei of all transcriptionally active cells throughout Drosophila development. This is consistent with a general role of PABP2 in mRNA polyadenylation. Analysis of Drosophila PABP2 function in a reconstituted mammalian polyadenylation system shows that the protein has the same functions as its bovine homolog in vitro : it stimulates poly(A) polymerase and is able to control poly(A) tail length.

Recognition of exonic splicing enhancer sequences by the Drosophila splicing repressor RSF1.

The Drosophila repressor splicing factor 1 (RSF1) comprises an N-terminal RNA-binding region and a C-terminal domain rich in glycine, arginine and serine residues, termed the GRS domain. Recently, RSF1 has been shown to antagonize splicing factors of the serine/arginine-rich (SR) family and it is, therefore, expected to play a role in processing of a subset of Drosophila pre-mRNAs through specific interactions with RNA. To investigate the RNA-binding specificity of RSF1, we isolated RSF1-binding RNAs using an in vitro selection approach. We have identified two RNA target motifs recognized by RSF1, designated A (CAACGACGA)- and B (AAACGCGCG)-type sequences. We show here that the A-type cognate sequence behaves as an SR protein-dependent exonic splicing enhancer. Namely, three copies of the A-type ligand bind SR proteins, stimulate the efficiency of splicing of reporter pre-mRNAs several fold and lead to inclusion of a short internal exon both in vitro and in vivo. However, three copies of a B-type ligand were much less active. The finding that RSF1 acts as a potent repressor of pre-mRNA splicing in vitro led us to propose that the equilibrium between a limited number of structurally-related general splicing activators or repressors, competing for common or promiscuous binding sites, may be a major determinant of the underlying mechanisms controlling many alternative pre-mRNA process-ing events.

Antagonism between RSF1 and SR proteins for both splice-site recognition in vitro and Drosophila development.

Specific recognition of splice sites within metazoan mRNA precursors (pre-mRNAs) is a potential stage for gene regulation by alternative splicing. Splicing factors of the SR protein family play a major role in this regulation, as they are required for early recognition of splice sites during spliceosome assembly. Here, we describe the characterization of RSF1, a splicing repressor isolated from Drosophila, that functionally antagonizes SR proteins. Like the latter, RSF1 comprises an amino-terminal RRM-type RNA-binding domain, whereas its carboxy-terminal part is enriched in glycine (G), arginine (R), and serine (S) residues (GRS domain). RSF1 induces a dose-sensitive inhibition of splicing for several reporter pre-mRNAs, an inhibition that occurs at the level of early splicing complexes formation. RSF1 interacts, through its GRS domain, with the RS domain of the SR protein SF2/ASF and prevents the latter from cooperating with the U1 small nuclear ribonucleoprotein particle (U1 snRNP) in binding pre-mRNA. Furthermore, overproduction of RSF 1 in the fly rescues several developmental defects caused by overexpression of the splicing activator SR protein B52/ SRp55. Therefore, RSF1 may correspond to the prototypical member of a novel family of general splicing repressors that selectively antagonize the effect of SR proteins on 5' splice-site recognition.

The Drosophila modifier of variegation modulo gene product binds specific RNA sequences at the nucleolus and interacts with DNA and chromatin in a phosphorylation-dependent manner.

modulo belongs to the modifier of Position Effect Variegation class of Drosophila genes, suggesting a role for its product in regulating chromatin structure. Genetics assigned a second function to the gene, in protein synthesis capacity. Bifunctionality is consistent with protein localization in two distinct subnuclear compartments, chromatin and nucleolus, and with its organization in modules potentially involved in DNA and RNA binding. In this study, we examine nucleic acid interactions established by Modulo at nucleolus and chromatin and the mechanism that controls the distribution and balances the function of the protein in the two compartments. Structure/function analysis and oligomer selection/amplification experiments indicate that, in vitro, two basic terminal domains independently contact DNA without sequence specificity, whereas a central RNA Recognition Motif (RRM)-containing domain allows recognition of a novel sequence-/motif-specific RNA class. Phosphorylation moreover is shown to down-regulate DNA binding. Evidence is provided that in vivo nucleolar Modulo is highly phosphorylated and belongs to a ribonucleoprotein particle, whereas chromatin-associated protein is not modified. A functional scheme is finally proposed in which modification by phosphorylation modulates Mod subnuclear distribution and balances its function at the nucleolus and chromatin.

Phosphorylation of the Drosophila engrailed protein at a site outside its homeodomain enhances DNA binding.

The engrailed gene encodes a homeodomain-containing phosphoprotein that binds DNA. Here, we show that engrailed protein is posttranslationally modified in embryos and in embryo-derived cultured cells but is essentially unmodified when expressed in Escherichia coli. Engrailed protein produced by bacteria can be phosphorylated in nuclear extracts prepared from Drosophila embryos, and phosphotryptic peptides from this modified protein partly reproduce two-dimensional maps of phosphotryptic fragments obtained from metabolically labeled engrailed protein. The primary embryonic protein kinase modifying engrailed protein is casein kinase II (CK-II). Analysis of mutant proteins revealed that the in vitro phosphoacceptors are mainly clustered in a region outside the engrailed homeodomain and identified serines 394, 397, 401, and 402 as the targets for CK-II phosphorylation. CK-II-dependent phosphorylation of an N-truncated derivative of engrailed protein purified from bacteria increased its DNA binding 2-4-fold.

Novel Drosophila melanogaster genes encoding RRM-type RNA-binding proteins identified by a degenerate PCR strategy.

We are interested in identifying Drosophila melanogaster RNA-binding proteins involved in important developmental decisions made at the level of mRNA processing, stability, localization or translational control. A large subset of the proteins known to interact with specific RNA sequences shares an evolutionarily conserved 80-90-amino-acid (aa) domain referred to as an RNA-recognition motif (RRM), including two ribonucleoprotein identifier sequences known as RNP-1 and RNP-2. Hence, we have herein applied degenerate polymerase chain reaction (PCR) methodology to clone three additional members (termed rox2, rox8 and rox21) of the D. melanogaster RRM-protein gene superfamily encoding putative trans-acting regulatory factors. Representative cDNA clones were isolated, the conceptual aa sequences of the candidate Rox proteins were inferred from their nucleotide sequences, and database searches were conducted. Rox2 displays extensive aa sequence similarities to putative RNA-binding proteins encoded by the genomes of the plants Oryza sativa and Arabidopsis thaliana; Rox21 resembles essential metazoan pre-mRNA splicing factors; as described elsewhere, Rox8 is likely a fly homolog of the two human TIA-1-type nucleolysins [Brand and Bourbon, Nucleic Acids Res. 21 (1993) 3699-3704].

The developmentally-regulated Drosophila gene rox8 encodes an RRM-type RNA binding protein structurally related to human TIA-1-type nucleolysins.

We report the molecular analysis of a novel Drosophila melanogaster gene, rox8, isolated in a PCR-based screen for sequences encoding RRM-type RNA-binding polypeptides. The rox8 gene is predicted to encode a 50-kilodalton protein displaying extensive amino acid sequence similarities (46% overall identity; 57 to 60% similarity) to the two recently described human TIA-1-type nucleolysins. These cytolytic granule associated proteins, which bind polyadenylated sequences in vitro and trigger DNA fragmentation in permeabilized target cells, are suspected to participate in the apoptotic cell death pathway induced by T-lymphocytes and natural killer cells. The structural relatedness of the three proteins includes three tandemly-repeated consensus RNA-recognition motifs at the N-terminal end and a putative membrane targeting signal at the C-terminal end. rox8 cytologically maps to 95D5-9 on the right arm of the third chromosome. Two rox8 transcripts of 3 and 3.3 kb in length, respectively, result from a developmentally-modulated alternative usage of different polyadenylation sites and are differentially accumulated throughout the fly life cycle. Molecular characterization of rox8 represents the first step in a genetic analysis of the potential roles of a TIA-1-related protein in RNA metabolism and/or programmed cell death in Drosophila.

Nucleolin gene organization in rodents: highly conserved sequences within three of the 13 introns.

The complete nucleotide (nt) sequence of the rat nuc gene encoding nucleolin, the major nucleolar-specific protein in eukaryotic exponentially growing cells, is compared with the corresponding locus recently characterized in mouse. [Bourbon et al., J. Mol. Biol. 200 (1988) 627-638]. In both murine species the genomic organization has been strikingly conserved during evolution, i.e., the coding region extends over 9 kb and is split into 14 exons, encoding a 712-amino acid protein. Moreover, all the exon-intron junction positions were strictly maintained during evolution. More unexpectedly, this analysis revealed that several introns contain highly conserved sequence elements of about 120 nt. The nt sequence of the homologous locus isolated from a Chinese hamster genomic clone established that these regions were under unusually high selective constraints (84-96% identity between the hamster and murine nuc genes) and, although they do not contain open reading frames, they surprisingly appear to be more conserved than most of the exons, suggesting that they play an important role. Such an element of 130 nt presents features of known genes transcribed by RNA polymerase III. Furthermore, in the rat nuc pre-mRNA the 5'- and 3'-end regions of the last intron are fully complementary over 16 nt, and so are predicted to be included in a prominent stem structure. Moreover, an homologous RNA stem structure can be derived from the mouse sequence, including two compensatory nt changes. As the secondary structure would occlude the canonical sequences required for the proper excision of this intron in both murine species, this remarkable finding could be relevant to the regulation of the nuc gene expression at the RNA processing level.

Sequence and structure of the nucleolin promoter in rodents: characterization of a strikingly conserved CpG island.

We report the isolation of the complete genes encoding nucleolin from rat and hamster. The DNA clones were obtained from partial genomic libraries by probing with a genomic DNA fragment containing the leader and promoter regions of the mouse nucleolin gene. We have determined the complete nucleotide sequence of the 5'-terminal region for the three rodent species. The sequenced regions extend over 1 kb downstream and upstream from the cap sites and include a conserved CpG island 1500 nucleotides (nt) long. The 5' end of the CpG island in each species has maintained a long alternating purine-pyrimidine sequence which could adopt a Z-DNA conformation. By sequence comparison, 42 blocks of homology are defined in the 5'-terminal region, of which 36 appear in the CpG island and contain numerous conserved CpG dinucleotides. Two blocks, 110 and 49 nt long, encompassing the cap sites and the region immediately upstream, respectively, present features characteristic of regulated genes: a possible TATA box (ATTA), two pyrimidine-rich nucleotide stretches and two inverted juxtaposed CCAAT-like boxes (GGTTGG). Furthermore, the adjacent upstream conserved region presents features characteristic of housekeeping genes: four G/C boxes, embedded in a high G + C-content sequence, among them one presenting a perfect consensus Sp 1-binding site (GCCCCGCCCC). Among unusual features, we report numerous large G + C-rich conserved sequences located in the first intron. One of these sequences contains two G/C boxes which border a sequence presenting a dyad symmetry (GCGCACGTGCTC). Our findings shed some light on the putative role of the CpG island. We show that CpG-rich sequence motifs are under strong selective pressure over the whole 5'-terminal region and are presumably involved in regulatory mechanisms.

Structure of the mouse nucleolin gene. The complete sequence reveals that each RNA binding domain is encoded by two independent exons.

Nucleolin is a multifunctional nucleolar protein involved in the synthesis, packaging and maturation of pre-rRNA in eukaryotic cells. We describe the molecular organization and complete sequence of the mouse nucleolin gene, the first higher eukaryotic gene encoding a protein that is both an RNA binding protein involved in rRNA processing and a specific nucleolar protein. The nucleolin gene extends over 9000 base-pairs and is split into 14 exons that encode the 706 amino acid residues of the protein. The promoter sequence is G + C-rich (67% G + C) with four G/C boxes, it lacks bona fide TATA and CAAT boxes and shows capping site heterogeneity. The existence of pyrimidine-rich motifs, similar to those found in the promoter of ribosomal protein genes, could be relevant to the co-regulation of genes whose products are involved in ribosome biogenesis. Nucleolin contains four RNA binding domains, each about 80 amino acid residues long, which include the 11-residue core ribonucleoprotein consensus motif. Each domain is encoded by two exons, with an intervening sequence interrupting the conserved core motif at roughly the same amino acid position. This latter result suggests that the RNA binding domains are composed of two independent subdomains, whose functions remain to be determined.

Maturation of a 100 kDa protein associated with preribosomes in Chinese hamster ovary cells.

A 100 kDa nucleolar protein which is transitorily associated with preribosomes in the nucleoli of Chinese hamster ovary cells has been found to be specifically cleaved by a thiol protease. During an 'in vitro' incubation of nucleoli, the 100 kDa protein is processed into eight different proteins which are detected by immunoreaction with a serum raised against the 100 kDa protein. Qualitative and quantitative variations in the maturation products of the 100 kDa protein are obtained by 'in vitro' incubation of the 60S and 80S preribosomes. The 100 kDa protein has been purified to homogeneity with the protease activity still associated. The properties of the enzyme are described and its role in the maturation of preribosomes is discussed.