Function of RRM domains of Drosophila melanogaster ELAV: Rnp1 mutations and rrm domain replacements with ELAV family proteins and SXL

Members of the ELAV family of proteins contain three RNA recognition motifs (RRMs), which are highly conserved. ELAV, a Drosophila melanogaster member of this family, provides a vital function and exhibits a predominantly nuclear localization. To investigate if the RNA-binding property of each of the ELAV RRMs is required for ELAV's in vivo function, amino acid residues critical in RNA binding for each RRM were individually mutated. A stringent genetic complementation test revealed that when the mutant protein was the sole source of ELAV, RNA-binding ability of each RRM was essential to ELAV function. To assess the degree to which each domain was specific for ELAV function and which domains perhaps performed a function common to related ELAV proteins, we substituted an ELAV RRM with the corresponding RRM from RBP9, the D. melanogaster protein most homologous to ELAV; HuD, a human ELAV family protein; and SXL, which, although evolutionarily related, is not an ELAV family member. This analysis revealed that RRM3 replacements were fully functional, but RRM1 and RRM2 replacements were largely nonfunctional. Under less stringent conditions RRM1 and RRM2 replacements from SXL and RRM1 replacement from RBP9 were able to provide supplemental function in the presence of a mutant hypomorphic ELAV protein.     

Evidence for 3' untranslated region-dependent autoregulation of the Drosophila gene encoding the neuronal nuclear RNA-binding protein ELAV.

The Drosophila locus embryonic lethal abnormal visual system (elav) encodes a nuclear RNA-binding protein essential for normal neuronal differentiation and maintenance of neurons. ELAV is thought to play its role by binding to RNAs produced by other genes necessary for neuronal differentiation and consequently to affect their metabolism by an as yet unknown mechanism. ELAV structural homologues have been identified in a wide range of organisms, including humans, indicating an important conserved role for the protein. Analysis of elav germline transformants presented here shows that one copy of elav minigenes lacking a complete 3'' untranslated region (3'' UTR) rescues null mutations at elav, but that two copies are lethal. Additional in vivo experiments demonstrate that elav expression is regulated through the 3'' UTR of the gene and indicate that this level of regulation is dependent upon ELAV itself. Because ELAV is an RNA-binding protein, the simplest model to account for these findings is that ELAV binds to the 3'' UTR of its own RNA to autoregulate its expression. I discuss the implications of these results for normal elav function.     

Promoter analysis of the Drosophila melanogaster gene encoding the pterin 4alpha-carbinolamine dehydratase.

Pterin-4alpha-carbinolamine dehydratase (PCD) is a key enzyme in the regeneration pathway of tetrahydrobiopterin. Previously, we isolated and reported the Drosophila melanogaster gene encoding PCD. In the present study, we isolated and characterized the Drosophila virilis gene encoding PCD. The Drosophila virilis PCD gene has two introns and an open reading frame to encode a protein of 101 amino acids. The amino acid sequence of Drosophila virilis PCD shows a 83% homology to that of the Drosophila melanogaster PCD protein. From the alignment of the nucleotide sequence in the 5'-flanking region of the Drosophila melanogaster and Drosophila virilis PCD genes, we found four conserved sequences. Using a transient transfection assay, we showed that one of the conserved sequences (-127 to approximately -115) is critical for expression, also the minimal promoter region between -127 and +51 is necessary for the efficient expression of Drosophila melanogaster PCD.     

Nucleotide variation at the no-on-transient A gene in Drosophila littoralis

The no-on-transient A (nonA) gene encodes a putative RNA-binding protein, and mutations in this gene are known to affect vision, male courtship song and viability in Drosophila melanogaster. Here we have sequenced the coding region of the nonA gene of Drosophila littoralis and compared it with those of Drosophila virilis and D. melanogaster. All portions of nonA appeared to be conserved between D. littoralis and D. virilis, while the 5' region of the gene of these two species showed high divergence from that of a more distantly-related species, D. melanogaster. The same was true for the glycine repeat regions. No significant deviation from neutrality was observed in the analysis of intraspecific nucleotide variation in 5' or 3' region of the nonA gene in D. littoralis population. Also, comparison of D. littoralis sequences with homologous sequence of D. virilis suggests that the gene is evolving neutrally in D. virilis group. Divergence of the 5' regions between D. virilis group species and D. melanogaster could be a result of positive selection, but this finding is obscured by the long divergence time of the species groups.     

A cytosolic form of aminopeptidase P from Drosophila melanogaster: molecular cloning and characterization

Using a functional genomic approach, we have identified and characterized a cytosolic form of aminopeptidase P from Drosophila melanogaster. This study represents the first characterization of an insect aminopeptidase P. The complete sequence of a 12.5 kbp genomic clone from D. melanogaster showed the presence of a 1,839 bp ORF, encoding a protein of 613 amino acids with a calculated molecular mass of 68.5 kDa. The deduced amino acid sequence was 48% identical and 66% similar to rat and human cytosolic aminopeptidase P. Amino acids important for catalytic activity and the metal binding ligands were found to be conserved between Drosophila AP-P and its mammalian homologues. The recombinant enzyme expressed in Escherichia coli hydrolyzed the amino terminal Xaa-Pro bond of substance P and bradykinin, revealing its functional identity. Further enzyme characterization showed the enzyme to be a manganese-dependent metallopeptidase. Immunoblot analysis showed that DAP-P is located exclusively in the cytosol and is temporally regulated during Drosophila development. In the adult fly, the protein could be detected in gut, testis and ovary, with a high level of expression in brain.     

Retrotransposon gtwin: structural analysis and distribution in Drosophila melanogaster strains

A search for noncanonical variants of the gypsy retrotransposon (MDG4) in the genome of the Drosophila melanogaster strain G32 led to the cloning of four copies of the poorly studied 7411-bp gtwin element. Sequence analysis showed that gtwin belongs to a family of endogeneous retroviruses, which are widespread in the Drosophila genome and have recently been termed insect erantiviruses. The gtwin retrotransposon is evolutionarily closest to MDG4, as evident from a good alignment of their nucleotide sequences including ORF1 (the pol gene) and ORF3 (the env gene), as well as the amino acid sequences of their protein products. These regions showed more than 75% homology. The distribution of gtwin was studied in several strains of the genus Drosophila. While strain G32 contained more than 20 copies of the element, ten other D. melanogaster strains carried gtwin in two to six copies per genome. The gtwin element was not detected in D. hydei or D. virilis. Comparison of the cloned gtwin sequences with the gtwin sequence available from the D. melanogaster genome database showed that the two variants of the mobile element differ by the presence or absence of a stop codon in the central region of ORF3. Its absence from the gtwin copies cloned from the strain G32 may indicate an association between the functional state of ORF3 and amplification of the element.     

Patterns of nucleotide polymorphism and divergence in the odorant-binding protein genes OS-E and OS-F: analysis in the melanogaster species subgroup of Drosophila

The Olfactory Specific-E and -F genes (OS-E and OS-F) belong to the odorant-binding protein gene family, which includes the general odorant-binding proteins and the pheromone-binding proteins. In Drosophila melanogaster, these genes are arranged in tandem in a genomic region near the centromere of chromosome arm 3R. We examined the pattern of DNA sequence variation in an approximately 7-kb genomic region encompassing the two OS genes in four species of the melanogaster subgroup of Drosophila and in a population sample of D. melanogaster. We found that both the OS-E and the OS-F gene are present in all surveyed species. Nucleotide divergence estimates would support that the two genes are functional, although they diverge in their functional constraint. The pattern of nucleotide variation in D. melanogaster also differed between genes. Variation in the OS-E gene region exhibited an unusual and distinctive pattern: (i) a relatively high number of fixed amino acid replacements in the encoded protein and (ii) a peak of nucleotide polymorphism around the OS-E gene. These results are unlikely under the neutral model and suggest the action of natural selection in the evolution of the two odorant-binding protein genes.     

Conservation of gene order, structure and sequence between three closely linked genes in Drosophila melanogaster and Drosophila virilis

In Drosophila melanogaster, the apparently unrelated genes anon-66Da, RpL14, and anon-66Db (from telomere to centromere) are located on a 5547 bp genomic fragment on chromosome arm 3L at cytological position 66D8. The three genes are tightly linked, and flanked by two relatively large genes with unknown function. We have taken a comparative genomic approach to investigate the evolutionary history of the three genes. To this end we isolated a Drosophila virilis 7.3 kb genomic fragment which is homologous to a 5.5 kb genomic region of D. melanogaster. Both fragments map to Muller's element D, namely to section 66D in D. melanogaster and to section 32E in D. virilis, and harbor the genes anon-66Da, RpL14, and anon-66Db. We demonstrate that the three genes exhibit a high conservation of gene topography in general and in detail. While most introns and intergenic regions reveal sequence divergences, there are, however, a number of interspersed conserved sequence motifs. In particular, two introns of the RpL14 gene contain a short, highly conserved 60 nt long sequence located at corresponding positions. This sequence represents a novel Drosophila small nucleolar RNA, which is homologous to human U49. Whereas DNA flanking the three genes shows no significant interspecies homologies, the 3'-flanking region in D. virilis contains sequences from the transposable element Penelope. The Penelope family of transposable elements has been shown to promote chromosomal rearrangements in the D. virilis species group. The presence of Penelope sequences in the D. virilis 7.3 kb genomic fragment may be indicative for a transposon-induced event of transposition which did not yet scramble the order of the three genes but led to the breakdown of sequence identity of the flanking DNA.     

Association of the neuron-specific RNA binding domain-containing protein ELAV with the coiled body inDrosophila neurons

The subcellular distribution of theDrosophila nervous system-specific RNA binding domain-containing protein ELAV was investigated using ELAV-specific antibodies and scanning confocal laser microscopy. ELAV is predominantly localized within the nucleus where it concentrates within discrete domains we describe as dots and webs. To characterize these discrete domains an analysis ofDrosophila coiled bodies was initiated. The polyclonal antibody R288 raised against human coilin was used to identify coiled bodies in cells of theDrosophila larval central nervous system. Double-labeling immunohistochemistry showed that, similar to vertebrate and plant systems, small nuclear ribonucleo-proteins are enriched within these structures. Further analysis of ELAV revealed that subnuclear domains enriched with this molecule localize within and close to coiled bodies and close to subnuclear domains enriched with splicing factors. A preliminary analysis aimed at defining a region within ELAV that may mediate a molecular or functional interaction important for its subnuclear localization revealed that deletion of the ELAV alanine/glutamine-rich amino-terminal auxiliary domain has no discernible effect on localization and that proteins produced fromelav lethal alleles distribute normally. Edited by: G. Dreyfuss     

Interspecific comparison of the period gene of Drosophila reveals large blocks of non-conserved coding DNA.

We have cloned and sequenced the coding region of the period (per) gene from Drosophila pseudoobscura and D. virilis. A comparison with that of D. melanogaster reveals that the conceptual translation products consist of interspersed blocks of conserved and non-conserved amino acid sequence. The non-conserved portion, comprising approximately 33% of the protein sequence, includes the perfect Thr-Gly repeat of D. melanogaster, which is absent from the D. pseudoobscura and D. virilis proteins. Based on these observations and cross-species transformation experiments, we suggest that the interspecific variability in the per primary amino acid sequence contributes to the control of species-specific behaviors.     

Evidence for selection at the fused locus of Drosophila virilis

The genomic DNA sequence of a 2.4-kb region of the X-linked developmental gene fused was determined in 15 Drosophila virilis strains. One common replacement polymorphism is observed, where a negatively charged aspartic amino acid is replaced by the noncharged amino acid alanine. This replacement variant is located within the serine/threonine kinase domain of the fused gene and is present in approximately 50% of the sequences in our sample. Significant linkage disequilibrium is detected around this replacement site, although the fused gene is located in a region of the D. virilis X chromosome that seems to experience normal levels of recombination. In a 600-bp region around the replacement site, all eight alanine sequences are identical; of the six aspartic acid sequences, three are also identical. The occurrence of little or no variation within the aspartic acid and alanine haplotypes, coupled with the presence of several differences between them, is very unlikely under the usual equilibrium neutral model. Our results suggest that the fused alanine haplotypes have recently increased in frequency in the D. virilis population.     

Larval salivary gland secretion proteins in Drosophila structural analysis of the Sgs-5 gene

The structure of the Drosophila melanogaster salivary gland secretion gene Sgs-5 has been determined by DNA sequence analysis of cloned genomic DNA. This developmentally and tissue-specific gene is a member of the third instar intermolt gene set and is under control of the insect molting hormone ecdysterone. RNA protection experiments show that the RNA coding region of Sgs-5 contains 769 nucleotides and is divided into three exons by two small introns. The protein-coding region appears to begin after a short untranslated RNA leader (33 nucleotides) and to result in a protein of 163 amino acids. The first 18 amino acids give the amino-terminal end the highly hydrophobic nature characteristic of a signal peptide.     

Conserved regions of the timeless (tim) clock gene in Drosophila analyzed through phylogenetic and functional studies.

Circadian (approximately 24-hr) rhythms in Drosophila melanogaster depend upon cyclic expression of the period (per) and timeless (tim) genes, which encode interacting components of the endogenous clock. The per gene has been isolated from other insects and, more recently, a per ortholog was found in mammals where its expression oscillates in a circadian fashion. We report here the complete sequence of a tim gene from another species, Drosophila virilis. TIM is better conserved than the PER protein is between these two species (76 vs. 54% overall amino acid identity), and putative functional domains, such as the PER interaction domains and the nuclear localization signal, are highly conserved. The acidic domain and the cytoplasmic localization domain, however, are within the least conserved regions. In addition, the initiating methionine in the D. virilis gene lies downstream of the proposed translation start for the original D. melanogaster tim cDNA and corresponds to the one used by D. simulans and D. yakuba. Among the most conserved parts of TIM is a region of unknown function near the N terminus. We show here that deletion of a 32 amino acid segment within this region affects rescue of rhythms in arrhythmic tim01 flies. Flies carrying a full-length tim transgene displayed rhythms with approximately 24-hr periods, indicating that a fully functional clock can be restored in tim01 flies through expression of a tim transgene. Deletion of the segment mentioned above resulted in very long activity rhythms with periods ranging from 30.5 to 48 hr.