spindle E/homeless

EVOLUTIONARY HOMOLOGS part 1/2

Drosophila helicases

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 the 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).

In Drosophila melanogaster, position-effect variegation of the white gene has provided a useful phenomenon by which to study chromosome structure and the genes that modify it. A new enhancer of variegation locus, Helicase at 25E, has been discovered in Drosophila. Deletion of the mutation Helicase at 25E enhances white variegation; this can be reversed by a transformed copy of Helicase at 25E+. In the presence of two endogenous copies, the transformed Helicase at 25E+ behaves as a suppressor of variegation. Helicase at 25E is an essential gene and functions both maternally and zygotically. The Helicase at 25E protein is similar to known RNA helicases, but contains an unusual variant (DECD) of the DEAD motif common to these proteins. Potential Helicase at 25E homologs have been found in mammals, yeast and worms. Helicase at 25E protein associates with salivary gland chromosomes and locates to nuclei of embryos and ovaries, but disappears in mitotic domains of embryos as chromosomes condense. It is proposed that the Helicase at 25E protein promotes an open chromatin structure that favors transcription during development by regulating the spread of heterochromatin, and that Helicase at 25E is regulated by, and may have a role in, the mitotic cell cycle during embryogenesis (Eberl, 1997).

Yeast helicases: PRP2, PRP8 and PRP22

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. One such DEAH protein, PRP2 of S. cerevisiae plays a role 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 could account for the previously reported differences between the precatalytic, pre-mRNA-containing spliceosome and the "active," intermediate-containing spliceosome. The post-PRP2-ATP spliceosome was further isolated and found able to carry out the subsequent reaction apparently in the absence of PRP2 and ATP. It is hypothesized that PRP2 functions as a molecular motor, similar to some DExH ATPases in transcription, in the activation of the precatalytic spliceosome for the transesterification reaction (Kim, 1996).

The RNA helicase-like splicing factor PRP2 interacts only transiently with spliceosomes. To facilitate analysis of PRP2 interactions with spliceosomal components, PRP2 protein was stalled in splicing complexes using two different methods. A dominant negative mutant form of PRP2 protein, which associates stably with spliceosomes, was found to interact directly with pre-mRNAs, as demonstrated by UV-crosslinking experiments. The use of various mutant and truncated pre-mRNAs revealed that this interaction requires a spliceable pre-mRNA and an assembled spliceosome; a 3' splice site is not required. To extend these observations to the wild-type PRP2 protein, spliceosomes were depleted of ATP; PRP2 protein interacts with pre-mRNA in these spliceosomes in an ATP-independent fashion. Comparison of RNA binding by PRP2 protein in the presence of ATP or gamma S-ATP shows that ATP hydrolysis rather than mere ATP binding is required to release PRP2 protein from pre-mRNA. Since PRP2 is an RNA-stimulated ATPase, these experiments strongly suggest that the pre-mRNA is the native co-factor stimulating ATP hydrolysis by PRP2 protein in spliceosomes. Since PRP2 is a putative RNA helicase, it is proposed that the pre-mRNA is the target of RNA displacement activity of PRP2 protein, promoting the first step of splicing (Teigelkamp, 1994).

To characterize sequences in the RNA helicase-like PRP2 protein of Saccharomyces cerevisiae that are essential for its function in pre-mRNA splicing, a pool of random PRP2 mutants was generated. A dominant negative allele was isolated which, when overexpressed in a wild-type yeast strain, inhibits cell growth by causing a defect in pre-mRNA splicing. This defect is partially alleviated by simultaneous co-overexpression of wild-type PRP2. The dominant negative PRP2 protein inhibits splicing in vitro and causes the accumulation of stalled splicing complexes. Immunoprecipitation with anti-PRP2 antibodies confirms that dominant negative PRP2 protein competes with its wild-type counterpart for interaction with spliceosomes, with which the mutant protein remains associated. The PRP2-dn1 mutation leads to a single amino acid change within the conserved SAT motif that in the prototype helicase eIF-4A is required for RNA unwinding. Purified dominant negative PRP2 protein has approximately 40% of the wild-type level of RNA-stimulated ATPase activity. Because ATPase activity is reduced only slightly, but splicing activity is abolished, it is proposed that the dominant negative phenotype is due primarily to a defect in the putative RNA helicase activity of PRP2 protein (Plumpton, 1994).

Five small nuclear RNAs (snRNAs) are required for nuclear pre-messenger RNA splicing: U1, U2, U4, U5 and U6. The yeast U1 and U2 snRNAs base-pair (respectively) to the 5' splice site and branch-point sequences of introns. The role of the U5 and U4/U6 small nuclear ribonucleoprotein particles (snRNPs) in splicing is not clear, though a catalytic role for the U6 snRNA has been proposed. Less is known about yeast splicing factors, but the availability of genetic techniques in Saccharomyces cerevisiae has led to the identification of mutants deficient in nuclear pre-mRNA splicing (prp2-prp27). Several PRP genes have now been cloned and their protein products characterized. The PRP8 protein is a component of the U5 snRNP and associates with the U4/U6 snRNAs/snRNP to form a multi-snRNP particle believed to be important for spliceosome assembly. Extragenic suppressors of the prp8-1 mutation of S. cerevisiae have been isolated. This study presents the preliminary characterization of one of these suppressors, spp81 is presented. The predicted amino-acid sequence of the SPP81 protein shows extensive similarity to a recently identified family of proteins thought to possess ATP-dependent RNA helicase activity. The possible role of this putative helicase in nuclear pre-mRNA splicing is discussed (Jamieson, 1991).

The product of the yeast PRP22 gene acts late in the splicing of yeast pre-messenger RNA, mediating the release of the spliced mRNA from the spliceosome. The predicted PRP22 protein sequence shares extensive homology with that of PRP2 and PRP16 proteins, which are also involved in nuclear pre-mRNA splicing. The homologous region contains sequence elements characteristic of several demonstrated or putative ATP-dependent RNA helicases. A putative RNA-binding motif originally identified in bacterial ribosomal protein S1 and Escherichia coli polynucleotide phosphorylase has also been found in PRP22 (Company, 1991).

Yeast helicases: Other DEAD-box 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 all 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), which 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 the 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 show 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 the 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. Rrp3 has been purified to homogeneity and has a weak RNA-dependent ATPase activity that is not specific for rRNA (O'Day, 1996).

Viral and vertebrate helicases

continued: Evolutionary homologs part 2/2 |

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