Interactive Fly, Drosophila



Drosophila helicases

The homeless gene of Drosophila is required for anteroposterior and dorsoventral axis formation during oogenesis. Females homozygous for mutations in hls occasionally, but infrequently, generate early egg chambers in which the oocyte is positioned incorrectly within the cyst. With high frequencies, late-stage egg chambers exhibit a ventralized chorion. Sequence analysis of the hls cDNA predicts a protein with amino-terminal homology to members of the DE-H family of RNA-dependent ATPases and putative helicases. Drosophila Maleless (required for dosage compensation) and two yeast splicing factors (PRP2 and PRP16) show a similarity of 51% in the amino-terminal third of Hls. To analyze Hls function, RNA localization patterns were determined for seven different transcripts in hls mutant ovaries. Previtellogenic transport to the oocyte is unaffected for all transcripts examined. Transport and localization of Bicoid and Oskar messages during vitellogenic stages are strongly disrupted, and the distribution and/or quantity of Gurken, Orb, and Fs(1)K10 mRNAs are also affected, but to a lesser degree. In contrast, Hu-li tai shao and Bicaudal-D transcripts are transported and localized normally in hls mutants. In addition, Kinesin heavy chain:beta-Galactosidase fusion protein fails to localize correctly to the posterior of the oocyte in vitellogenic egg chambers. Examination of the microtubule structure with anti-alpha-Tubulin antibodies reveals aberrant microtubule organizing center movement and an abnormally dense cytoplasmic microtubule meshwork (Gillespie, 1995).

Vasa protein is essential for the assembly of the pole plasm, a special cytoplasm found in the posterior portion of the egg and early embryo. Vasa is an RNA binding protein with an RNA dependent helicase. Vasa has been associated with two developmental processes. The first involves assembling the perinuclear region of the oocyte. Perinuclear cytoplasm is the precursor of the pole plasm. The helicase function of Vasa is required later, in a second process, for the assembly of pole plasm. Posterior localization of Vasa depends most likely on an interaction with Oskar. Oskar can successfully localize to the posterior pole without Vasa, but Oskar by itself cannot assemble the pole plasm. Both Oskar and Vasa activity are necessary for Nanos mRNA localization at the posterior pole (Liang, 1994). The function of VASA is to overcome the repressive effect of Nanos translational control element, an evolutionarily conserved dual stem-loop structure in the 3' untranslated region which acts independently of the localization signal to repress translation of Nanos mRNA (Gavis, 1996).

Vaccinia virus RNA polymerase terminates transcription in response to a specific signal UUUUUNU in the nascent RNA. Transduction of this signal to the elongating polymerase requires a trans-acting viral termination factor (VTF/capping enzyme), and is coupled to the hydrolysis of ATP. Recent studies suggest that ATP hydrolysis is catalyzed by a novel termination protein (factor X), which is tightly associated with the elongation complex. Factor X is identified as NPH-I (nucleoside triphosphate phosphohydrolase-I), a virus-encoded DNA-dependent ATPase of the DExH-box family. NPH-I serves two roles in transcription: (1) it acts in concert with VTF/CE to catalyze release of UUUUUNU-containing nascent RNA from the elongation complex, and (2) it acts by itself as a polymerase elongation factor to facilitate readthrough of intrinsic pause sites. A mutation (K61A) in the GxGKT motif of NPH-I abolishes ATP hydrolysis and eliminates the termination and elongation factor activities. Related DExH proteins may have similar roles at postinitiation steps during cellular mRNA synthesis. NPH-I is a prototype of a distinct subfamily of DExH proteins that includes Snf2 and its numerous homologs. These proteins are implicated in transcriptional activatin and repression in chromatin remodeling. Another protein, Drosophila Kc, has been found to suppress the appearance of long transcripts by promoting the release of nascent ranscripts from Pol II elongation complexes in an ATP-dependent manner (Xie, 1996). It may be worthwhile to consider that the positive and negative effects of some Snf2-like proteins on gene expression may be mediated by post-initiation steps of the transcription cycle (Deng, 1998).

Maleless homologs in other insects

In Drosophila melanogaster and Sciara ocellaris, dosage compensation occurs by hypertranscription of the single male X chromosome. This article reports the cloning and characterization in S. ocellaris of the gene homologous to maleless (mle) of D. melanogaster, which implements dosage compensation. The Sciara mle gene produces a single transcript, encoding a helicase, which is present in both male and female larvae and adults and in testes and ovaries. Both Sciara and Drosophila Mle proteins are highly conserved. The affinity-purified antibody to D. melanogaster Mle recognizes the S. ocellaris Mle protein. In contrast to Drosophila polytene chromosomes, where Mle is preferentially associated with the male X chromosome, in Sciara Mle is found associated with all chromosomes. Anti-Mle staining of Drosophila postblastoderm male embryos reveals a single nuclear dot, whereas Sciara male and female embryos present multiple intranuclear staining spots. This expression pattern in Sciara is also observed before blastoderm stage, when dosage compensation is not yet set up. The affinity-purified antibodies against D. melanogaster Msl1, Msl3, and Mof proteins involved in dosage compensation also reveal no differences in the staining pattern between the X chromosome and the autosomes in both Sciara males and females. These results have led to a proposal that different proteins in Drosophila and Sciara would implement dosage compensation (Ruiz, 2000).

Yeast helicases

The translation initiation factor eIF4E mediates the binding of the small ribosomal subunit to the cap structure at the 5' end of the mRNA. In Saccharomyces cerevisiae, the cap-binding protein eIF4E is mainly associated with eIF4G, forming the cap-binding complex eIF4F. Other proteins are detected upon purification of the complex on cap-affinity columns. Among them is p20, a protein of unknown function encoded by the CAF20 gene. p20 has a negative regulatory role in translational initiation. Deletion of CAF20 partially suppresses mutations in translation initiation factors. Overexpression of the p20 protein results in a synthetic enhancement of translation mutation phenotypes. Similar effects are observed for mutations in the DED1 gene, which has been isolated as a multicopy suppressor of a temperature-sensitive eIF4E mutation. The DED1 gene encodes a putative RNA helicase of the DEAD-box family. The analyses of its suppressor activity, of polysome profiles of ded1 mutant strains, and of synthetic lethal interactions with different translation mutants indicate that the Ded1 protein has a role in translation initiation in S. cerevisiae (de la Cruz, 1997).

The DED1 gene, which encodes a putative RNA helicase, has been implicated in nuclear pre-messenger RNA splicing in the yeast Saccharomyces cerevisiae. Translation, rather than splicing, is severely impaired in two newly isolated ded1 conditional mutants. Preliminary evidence suggests that the protein Ded1p may be required for the initiation step of translation, as is the distinct DEAD-box protein, eukaryotic initiation factor 4A (eIF4A). The DED1 gene could be functionally replaced by a mouse homolog, PL10, which suggests that the function of Ded1p in translation is evolutionarily conserved (Chuang, 1997).

In Saccharomyces cerevisiae, ribosomal biogenesis takes place primarily in the nucleolus, in which a single 35S precursor rRNA (pre-rRNA) is first transcribed and sequentially processed into 25S, 5.8S, and 18S mature rRNAs, leading to the formation of the 40S and 60S ribosomal subunits. Although many components involved in this process have been identified, an understanding of this important cellular process remains limited. One of the evolutionarily conserved DEAD-box protein genes in yeast, DBP3, is required for optimal ribosomal biogenesis. DBP3 encodes a putative RNA helicase, Dbp3p (523 amino acids in length) that bears a highly charged amino terminus consisting of 10 tandem lysine-lysine-X (KKX) repeats. Disruption of DBP3 is not lethal but yields a slow-growth phenotype. This genetic depletion of Dbp3p results in a deficiency of 60S ribosomal subunits and a delayed synthesis of the mature 25S rRNA, which is caused by a prominent kinetic delay in pre-rRNA processing at site A3 and to a lesser extent at sites A2 and A0. These data suggest that Dbp3p may directly or indirectly facilitate RNase MRP cleavage at site A3. The direct involvement of Dbp3p in ribosomal biogenesis is supported by the finding that Dbp3p is localized predominantly in the nucleolus. The (KKX) repeats are dispensable for Dbp3p's function in ribosomal biogenesis but are required for its proper localization. The (KKX) repeats thus represent a novel signaling motif for nuclear localization and/or retention (Weaver, 1997).

The phylogenetically conserved U14 small nucleolar RNA is required for processing of rRNA, and this function involves base pairing with conserved complementary sequences in 18S RNA. With a view to identifying other important U14 interactions, a stem-loop domain required for activity of Saccharomyces cerevisiae U14 RNAs (the Y domain) was first subjected to detailed mutational analysis. The mapping results showed that most nucleotides of the Y domain can be replaced without affecting function, except for loop nucleotides conserved among five different yeast species. Defective variants were then used to identify both intragenic and extragenic suppressor mutations. All of the intragenic mutations map within six nucleotides of the primary mutation, suggesting that suppression involves a change in conformation and that the loop element is involved in an essential intermolecular interaction rather than intramolecular base pairing. A high-copy extragenic suppressor gene, designated DBP4 (DEAD box protein 4), encodes an essential, putative RNA helicase of the DEAD-DEXH box family. Suppression by DBP4 restores the level of 18S rRNA and is specific for the Y domain but is not allele specific. DBP4 is predicted to function either in assembly of the U14 small nucleolar RNP or, more likely, in its interaction with other components of the rRNA processing apparatus. Mediating the interaction of U14 with precursor 18S RNA is an especially attractive possibility (Liang, 1997).

A new gene of S. cerevisiae, RRP3 (rRNA processing) is required for pre-rRNA processing. Rrp3 is a 60.9 kDa protein that is required for maturation of the 35S primary transcript of pre-rRNA and is required for cleavages leading to mature 18S RNA. RRP3 was identified in a PCR screen for DEAD box genes. DEAD box genes are part of a large family of proteins homologous to the eukaryotic transcription factor elF-4a. Most of these proteins are RNA-dependent ATPases and some of them have RNA helicase activity. This is the third yeast DEAD box protein that has been shown to be involved in rRNA assembly, but the only one required for the processing of 18S RNA. Mutants of the two other putative helicases, Spb4 and Drsl, both show processing defects in 25S rRNA maturation. In strains where Rrp3 is depleted, 35S precursor RNA is improperly processed. Cleavage normally occurs at sites A0O, Al and A2, but in the Rrp3 depletion stain cleavage occurs between A2 and B1. Rrp3 has been purified to homogeneity and has a weak RNA-dependent ATPase activity which is not specific for rRNA (O'Day, 1996).

In addition to small nuclear RNAs and spliceosomal proteins, ATP hydrolysis is needed for nuclear pre-mRNA splicing. A number of RNA-dependent ATPases that are involved in several distinct ATP-dependent steps in splicing have been identified in Saccharomyces cerevisiae and mammals. These so-called DEAD/H ATPases contain conserved RNA helicase motifs, although RNA unwinding activity has not been demonstrated in purified proteins. PRP2 of S. cerevisiae is involved in spliceosome activation. PRP2 binds to a precatalytic spliceosome prior to the first step of splicing. By blocking the activity of HP, a novel splicing factor(s) that is involved in a post-PRP2 step, it was found that PRP2 hydrolyzes ATP to cause a change in the spliceosome without the occurrence of splicing. The change is quite dramatic and can account for previously reported differences between the precatalytic, pre-mRNA-containing spliceosome and the "active," intermediate-containing spliceosome. The post-PRP2-ATP spliceosome has been further isolated and can carry out the subsequent reaction apparently in the absence of PRP2 and ATP. It is hypothesized that PRP2 functions as a molecular motor during transcription, similar to some DExH ATPases in the activation of the precatalytic spliceosome for the transesterification reaction (Kim 1996).

Vertebrate helicases

Continued maleless Evolutionary Homologs part 2/2

maleless: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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