Gene name - arrest
Synonyms - bruno (bru)
Cytological map position - 33D1--33D5
Function - mRNA binding protein
Symbol - aret
FlyBase ID: FBgn0000114
Genetic map position - 2-48
Classification - Ribonucleoprotein-type RNA-binding domain protein
Cellular location - cytoplasmic
Genes occasionally come to be known and referred to by more than one name. Such is the case with arrest, the more correct but less familiar term for the gene bruno. Bruno (Bru) is an ovarian and testicular messenger RNA-binding protein that regulates multiple mRNAs involved in female and male gametogenesis and is also active early in embryogenesis. Bruno functions in the translational repression of Oskar mRNA, and Bruno protein interacts physically with Vasa, an RNA helicase that is a positive regulator of Oskar translation. The gene arrest was characterized by Schupbach and Wieschaus (1991) as a female sterile mutation on the second chromosome of Drosophila. Bruno protein, later found to be identical to the protein coded for by arrest, is required for fertility in both sexes. Few germ cells are present in females that are hemizygous for strong alleles and OSK transcript is not detectable (Webster, 1997). Bruno is one of nearly a dozen maternal genes, belonging to the posterior group, which are involved directly or indirectly in assembly of the pole plasm.
Bruno was originally identified in UV cross-linking experiments as an ovarian protein that binds specfic sequences (Bruno response elements or BRE) in the 3' UTR of OSK mRNA (Kim-Ha, 1995). An expression screen based on the binding of Bru to its target sequence was designed in order to identify the gene coding for Bruno. An ovarian complementary DNA expression library was constructed in a plasmid vector and lysates of clones of transformed bacteria were examined for the presence of a protein that would specifically bind BRE+ mRNA. A plasmid encoding the binding activity was purified and used to clone the complete bruno gene (Webser, 1997).
Three distinct segments of OSK mRNA, termed A, B and C regions, contain Bruno-binding sites. The A and B regions are adjacent to one another near the beginning of the 3' UTR, whereas the C region is located downstream of the AB region, close to the polyadenylation site. The Bruno binding sites contain the consensus sequence UU(G/A)U(A.G)U(G/A)U. When this sequence is modified by mutation, Bru fails to bind. Oskar transgenes lacking Bru binding sites produce embryos that display substantial patterning defects. These defects indicate a posteriorization of the embryo and can be attributed to excess or mislocalized osk activity. These results suggest that Bruno normally acts to restrict OSK activity. BRE mutations have no effect on OSK mRNA localization; rather, they affect the level of translation. The posterior group genes cappuccino, spire, mago nashi, staufen and oo18, each of which are required for the localization of OSK mRNA to the posterior pole of the oocyte, are still required for OSK translation when Bruno-mediated translational repression is missing due to deleted BREs (Kim-Ha, 1995).
How does Bruno act to repress translation? It seems likely that although Bru binds OSK mRNA downstream of the protein coding region, it must affect either the initiation or the progression of translation. Two models have been proposed: the first involves the OSK polyadenylation [poly(A)] tail. Changes in poly(A) tail length are associated with changes in translation (see Drosophila Nanos for a discussion of the role of Nanos in Hunchback mRNA polyadenylation). This model predicts that an OSK mRNA mutant lacking BREs should have a longer poly(A) tail than that of wild-type transcript. However there is no obvous difference in tail length between normal and mutant OSK mRNAs. A second model hypothesizes an interaction between the 3' and 5' ends of the OSK message that influences translational initiation. It is possible that such an interaction promotes translational initiation and that Bruno interferes with this interaction.
What does the interaction between Bruno and Vasa protein signify? The fact that Vasa participates in the activation of OSK mRNA and that the requirement for Vasa is independent of the OSK 3'UTR suggests that Vasa does not activate OSK translation simply by relieving Bruno-mediated repression. Vasa may activate translation through interaction with the OSK 5' UTR. An interaction between Vasa and Bruno bound to the OSK 3' UTR might restrict the ability of Vasa to associate with the 5' UTR until after the OSK transcript is appropriately localized (Webster, 1997)
The arrest phenotype (female sterility) indicates that OSK mRNA cannot be the only target of Bruno regulation, since osk is not required for the early stages of oogenesis and the misregulaiton of OSK by mutation of the BREs does not cause defects in gametogenesis. In addition, although osk is expressed only in females, many arrest alleles are also male-sterile because of reduced numbers of sperm bundles and lack of motile sperm (Schupback, 1991 and Castrillon, 1993). Finally, the disrupted segmentation seen in embryos produced from arrestPA62/arrestPD41 transheterozygotes is not a phenotype that can be attributed to the misregulation of Oskar mRNA. Thus, Bruno carries out multiple roles in development; given its role in the repression of OSK mRNA translation, it is expected that Bru regulates the translation of multiple transcripts (Webster, 1997).
bruno codes for sex specific protein isoforms. There are three female-specific transcripts of 2.7, 3.3 and 3.7 kb, as well as a single male-specific transctipt of 4.0 kb. These transcripts are present in ovaries and testes, respectively, but are not detectable in the remaining somatic tissue. The three ovarian mRNAs are abundant in ovaries and in 0-2 hour embryos, but are extremely reduced or absent during the rest of embryogenesis and the larval stages of the cell cycle. Two pupal transcripts are evident. One migrates slightly slower than the male-specific message and one slightly faster than the 3.3-kb female specific message. The structural basis for these differences in mRNA sizes is not yet known. Testis extracts were assayed for the presence of a protein that binds Bruno response elements in mRNA. This protein is larger than the Bru protein found in ovary extracts. (Webster, 1997).
Male and female proteins differ only in the amino-terminal regions. The most notable features in the common portion of the proteins are three ribonucleoprotein-type RNA-binding domains: one at the C-terminus, and the other two adjacent to one another in the central portion of the protein. Database comparisons reveal that both the relative positioning and the sequence of the three Bru RNA-binding domains are highly conserved in evolution: they are homologous to those of human CUG-BP gene, as well as the Xenopus and Caenorhabditis elegans etr-1 genes. CUG-BP is identified as a protein that binds CUG repeats and may be involved in the human disease myotonic dystrophy. Xenopus etr-1 was isolated as a neural-specific marker (Webster, 1997 and references).
date revised: 25 January 98
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