Components Acting in Localization of Bicoid mRNA Are Conserved among Drosophila Species

Substantial insights into basic strategies for embryonic body patterning have been obtained from genetic analyses of Drosophila melanogaster. This knowledge has been used in evolutionary comparisons to ask if genes and functions are conserved. To begin to ask how highly conserved are the mechanisms of mRNA localization, a process crucial to Drosophila body patterning, we have focused on the localization of bcd mRNA to the anterior pole of the embryo. Here we consider two components involved in that process: the exuperantia (exu) gene, required for an early step in localization; and the cis-acting signal that directs bcd mRNA localization. First, we use the cloned D. melanogaster exu gene to identify the exu genes from Drosophila virilis and Drosophila pseudoobscura and to isolate them for comparisons at the structural and functional levels. Surprisingly, D. pseudoobscura has two closely related exu genes, while D. melanogaster and D. virilis have only one each. When expressed in D. melanogaster ovaries, the D. virilis exu gene and one of the D. pseudoobscura exu genes can substitute for the endogenous exu gene in supporting localization of bcd mRNA, demonstrating that function is conserved. Second, we reevaluate the ability of the D. pseudoobscura bcd mRNA localization signal to function in D. melanogaster. In contrast to a previous report, we find that function is retained. Thus, among these Drosophila species there is substantial conservation of components acting in mRNA localization, and presumably the mechanisms underlying this process.     

DNA sequence variation at a duplicated gene: excess of replacement polymorphism and extensive haplotype structure in the Drosophila melanogaster bicoid region

The bicoid (bcd) gene of Drosophila has played an important role in understanding the system of developmental regulatory genes that controls segmentation in the fruit fly. Several studies in Drosophila and closely related insects suggest that bcd may be the result of a gene duplication in the Dipteran lineage. In addition, the presence of a large, conserved secondary structure in the 3' untranslated region (UTR) makes the bcd gene a good candidate for studying compensatory evolution and the relationship between RNA secondary structure and patterns of standing variation in natural populations. Despite these interesting aspects, a population-level analysis has until now not been performed on bcd. In this study, DNA sequence variation was examined for a 4-kb region of the bcd gene, including a portion of the 5' UTR, the entire coding region, and the 3' UTR, for 25 Drosophila melanogaster isofemale lines from Zimbabwe and one allele from D. simulans. Statistical tests revealed a significant excess of replacement polymorphisms in the D. melanogaster lineage that are clustered in two putative linker regions of the Bicoid protein. This result is consistent with a relaxation of selective constraints in these regions. In addition, we found a distinct haplotype structure and a significantly smaller number of haplotypes than predicted by the standard neutral model. It is unlikely that the haplotype structure is maintained by epistatic selection acting on the secondary structure in the 3' UTR or by the association of the bcd gene with polymorphic inversions. Instead, our two main observations, namely the occurrence of a haplotype structure and the excess of replacement polymorphisms, may indicate that the selective history of this gene is rather complex, involving both the relaxation of purifying selection in some parts of the protein and the action of positive selection in other parts of the gene region.     

RNA-RNA interaction is required for the formation of specific bicoid mRNA 3'' UTR-STAUFEN ribonucleoprotein particles.

The formation of the anterior pattern of the Drosophila embryo is dependent on the localization of the mRNA of the morphogen Bicoid (bcd) to the anterior pole of the egg cell. Staufen protein (STAU) is required in a late step of the localization to anchor the bcd mRNA in the anterior cytoplasm. We have shown previously that endogenous STAU associates specifically with injected bcd mRNA 3'-untranslated region (UTR), resulting in the formation of characteristic RNA-protein particles that are transported along microtubules of the mitotic spindles in a directed manner. The regions recognized by STAU in this in vivo assay are predicted to form three stem-loop structures involving large double-stranded stretches. Here, we show that the STAU interaction requires a double-stranded conformation of the stems within the RNA localization signal. In addition, base pairing between two single-stranded loops plays a major role in particle formation. This loop-loop interaction is intermolecular, not intramolecular; thus dimers or multimers of the RNA localization signal must be associated with STAU in these particles. The bcd mRNA 3' UTR can also dimerize in vitro in the absence of STAU. Thus, in addition to RNA-protein interactions, RNA-RNA interaction might be involved in the formation of ribonucleoprotein particles for transport and localization.     

Divergence of the Yellow Gene between Drosophila Melanogaster and D. Subobscura: Recombination Rate, Codon Bias and Synonymous Substitutions

The yellow (y) gene maps near the telomere of the X chromosome in Drosophila melanogaster but not in D. subobscura. Thus the strong reduction in the recombination rate associated with telomeric regions is not expected in D. subobscura. To study the divergence of a gene whose recombination rate differs between two species, the y gene of D. subobscura was sequenced. Sequence comparison between D. melanogaster and D. subobscura revealed several elements conserved in noncoding regions that may correspond to putative cis-acting regulatory sequences. Divergence in the y gene coding region between D. subobscura and D. melanogaster was compared with that found in other genes sequenced in both species. Both, yellow and scute exhibit an unusually high number of synonymous substitutions per site (p(s)). Also for these genes, the extent of codon bias differs between both species, being much higher in D. subobscura than in D. melanogaster. This pattern of divergence is consistent with the hitchhiking and background selection models that predict an increase in the fixation rate of slightly deleterious mutations and a decrease in the rate of fixation of slightly advantageous mutations in regions with low recombination rates such as in the y-sc gene region of D. melanogaster.     

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.     

The Drosophila virilis dopa decarboxylase gene is developmentally regulated when integrated into Drosophila melanogaster.

The dopa decarboxylase gene (Ddc) has been isolated from Drosophila virilis and introduced into the germ-line of Drosophila melanogaster by P-element mediated transformation. The integrated gene is induced at the correct stages during development with apparently normal tissue specificity, indicating that cis-acting elements required for regulation are functionally conserved between the two species. A comparison of the DNA sequences from the 5' flanking regions reveals a cluster of small (8-16 bp) conserved sequence elements within 150 bp upstream of the RNA startpoint, a region required for normal expression of the D. melanogaster Ddc gene.     

Recognition and long-range interactions of a minimal nanos RNA localization signal element

Localization of nanos (nos) mRNA to the germ plasm at the posterior pole of the Drosophila embryo is essential to activate nos translation and thereby generate abdominal segments. nos RNA localization is mediated by a large cis-acting localization signal composed of multiple, partially redundant elements within the nos 3' untranslated region. We identify a protein of approximately 75 kDa (p75) that interacts specifically with the nos +2' localization signal element. We show that the function of this element can be delimited to a 41 nucleotide domain that is conserved between D. melanogaster and D. virilis, and confers near wild-type localization when present in three copies. Two small mutations within this domain eliminate both +2' element localization function and p75 binding, consistent with a role for p75 in nos RNA localization. In the intact localization signal, the +2' element collaborates with adjacent localization elements. We show that different +2' element mutations not only abolish collaboration between the +2' and adjacent +1 element but also produce long-range deleterious effects on localization signal function. Our results suggest that higher order structural interactions within the localization signal, which requires factors such as p75, are necessary for association of nos mRNA with the germ plasm.     

Nucleotide Variation and Divergence in the Histone Multigene Family in Drosophila Melanogaster

Nucleotide differences in the histone H3 gene family in Drosophila melanogaster were studied on three levels: (1) within a chromosome, (2) within a population and (3) between species (D. melanogaster and Drosophila simulans). The average difference within the H3 gene within a chromosome was 0.0040 per nucleotide site, about 52% of that within a population (0.0077). The proportion of divergent sites between the two species was 0.0575, which is about 8.5 times the difference within a species. The distribution of divergence between species was similar to that of variation within a species. Divergence and variation were noted to be greatest in the 3' noncoding region and least in the coding region. Values intermediate between these were found for the 5' noncoding region. Divergence and variation in silent sites exceeded those in the total coding region, thus indicating possible purifying selection for amino-acid-altering change. Phylogenetic relations among H3 genes and genetic differences on these three levels are evidence for the concerted evolution of the histone gene family. The molecular mechanism by which variation is produced and maintained is discussed.     

Conservation of alternative splicing and genomic organization of the myosin alkali light-chain (Mlc1) gene among Drosophila species

The Mlc1 gene of Drosophila melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. In larval muscle and tubular and abdominal muscles of adults, all of the six exons are included in the spliced mRNA, whereas, in the fibrillar indirect flight muscle of adult, exon 5 is excluded from the mRNA. We show that this tissue-specific pattern of alternative splicing of the Mlc1 pre-mRNA is conserved in D. simulans, D. pseudoobscura, and D. virilis. Isolation and sequencing of the Mlc1 genes from these three other Drosophila species have revealed that the overall organization of the genes is identical and that the genes have maintained a very high level of sequence identity within the coding region. Pairwise amino acid identities are 94%-99%, and there are no charge changes among the proteins. Total nucleotide divergence within the coding region of the four genes supports the accepted genealogy of these species, but the data indicate a significantly higher rate of amino acid replacement in the branch leading to D. pseudoobscura. A comparison of nucleotide substitutions in the coding portions of exon 5 and exon 6, which encode the alternative carboxyl termini of the two MLC1 isoforms, suggests that exon 5 is subject to greater evolutionary constraints than is exon 6. In addition to the coding sequences, there is significant sequence conservation within the 5' and 3' noncoding DNA and two of the introns, including one that flanks exon 5. These regions are candidates for cis- regulatory elements. Our results suggest that evolutionary constraints are acting on both the coding and noncoding sequences of the Mlc1 gene to maintain proper expression and function of the two MLC1 polypeptides.      

Redundant RNA recognition events in bicoid mRNA localization.

A cis-acting signal in the 3' UTR of the Drosophila bicoid mRNA directs both the transport of the mRNA from the nurse cells to the oocyte and its anterior localization within the oocyte. Here we demonstrate that the signal mediates redundant RNA recognition events, A and B, that initiate largely overlapping programs of mRNA localization during oogenesis. Recognition event A requires a region encompassing stem-loops IV/V of the predicted secondary structure, and can be eliminated by a single nucleotide mutation. Localization initiated through event B begins slightly later in oogenesis, and requires sequences that have not been narrowly defined. Using forms of the 3' UTR lacking this RNA recognition redundancy, we reexamine the roles of the swallow, staufen, and exuperantia genes, which are all required for normal bicoid mRNA localization. Our results reveal that exuperantia first becomes essential for localization at a time when well-defined microtubule tracks between the nurse cells and oocyte disappear. Thus, exuperantia may specifically facilitate a form of nurse cell-to-oocyte mRNA transport not dependent on the microtubule tracks.     

Duplicative and conservative transpositions of larval serum protein 1 genes in the genus Drosophila

Interspecific comparative molecular analyses of transposed genes and their flanking regions can help to elucidate the time, direction, and mechanism of gene transposition. In the Drosophila melanogaster genome, three Larval serum protein 1 (Lsp1) genes (alpha, beta and gamma) are present and each of them is located on a different chromosome, suggesting multiple transposition events. We have characterized the molecular organization of Lsp1 genes in D. buzzatii, a species of the Drosophila subgenus and in D. pseudoobscura, a species of the Sophophora subgenus. Our results show that only two Lsp1 genes (beta and gamma) exist in these two species. The same chromosomal localization and genomic organization, different from that of D. melanogaster, is found in both species for the Lsp1beta and Lsp1gamma genes. Overall, at least two duplicative and two conservative transpositions are necessary to explain the present chromosomal distribution of Lsp1 genes in the three Drosophila species. Clear evidence for implication of snRNA genes in the transposition of Lsp1beta in Drosophila has been found. We suggest that an ectopic exchange between highly similar snRNA sequences was responsible for the transposition of this gene. We have also identified the putative cis-acting regulatory regions of these genes, which seemingly transposed along with the coding sequences.