BIOLOGICAL OVERVIEW

Brain tumor (Brat) is one of three NHL domain proteins found in Drosophila (Arama, 2000; Sonoda, 2001). The family name derives from three of the founding members: NCL-1, HT2A, and LIN-41 (Slack, 1998). All three factors have ties to RNA metabolism: the nucleoli in Caenorhabditis elegans ncl-1 mutants are enlarged (Frank, 1998); HT2A was identified by virtue of interaction with the RNA-binding protein HIV Tat (Fridell, 1995), and posttranscriptional regulation of lin-29 mRNA is abrogated in lin-41 mutants (Slack, 2000). Little is known of the biological roles of other family members, and no direct molecular mechanism has been described previously for any NHL domain protein (including Brat). The NHL domain of Brat mediates its recruitment to the 3' UTR of Hunchback (HB) mRNA. Recruitment occurs through protein-protein interactions with RNA-bound Pumilio and Nanos; formation of the resulting quaternary complex is essential for translational control of HB. These results suggest a general mechanism by which other NHL domain proteins may act to control posttranscriptional gene expression (Sonoda, 2001).

Maternally derived HB mRNA is uniformly distributed throughout the embryo; the mRNA is translationally repressed in the posterior, giving rise to an anterior-to-posterior gradient of Hb protein. Failure of this repression results in the abnormal accumulation of Hb in the posterior, which inhibits abdominal segmentation. Two conserved RNA-binding proteins, Pumilio (Pum) and Nanos (Nos), are specifically required to repress HB translation. Pum, which is distributed uniformly throughout the embryo, is the founding member of a large family of RNA-binding proteins. Pum binds to 32 nucleotide sites in the 3' UTR of HB (Nos Response Elements, NREs) to regulate HB translation. Nos, which initially is distributed as a gradient emanating from the posterior pole of the embryo, contains a conserved zinc finger that mediates nonspecific RNA binding. Nos is selectively recruited into a ternary complex on HB mRNA by NRE-bound Pum. The mechanism by which the resulting Nos/Pum/NRE complex regulates translation is not yet understood, although deadenylation is thought to play a role (Sonoda, 2001 and references therein).

To identify targets or cofactors of the Nos/Pum/NRE ternary complex, a yeast 'four-hybrid' experiment was performed; a Gal4 activation domain fusion library was screened for proteins that interact with the ternary complex. The bait contained the RNA-binding domain of Pum, full-length Nos, and NRE-bearing RNA. As anticipated, factors that interact with individual components in isolation were identified. However, one factor, which proved to be a fragment of Brat, interacts only with the ternary complex and not with either Nos alone, Pum alone, or a Pum/NRE binary complex. Deletion analysis revealed that recruitment of Brat is dependent on the conserved carboxy-terminal domain of Nos that mediates its interaction with Pum on HB mRNA (Sonoda, 1999), and not the amino-terminal domain of Nos that mediates interaction with Cup during early oogenesis (Verrotti, 2000). Mutational analysis further showed that a fragment of Brat consisting of little more than the NHL domain is recruited to the ternary complex. Protein-protein interaction experiments show that Nanos and Pumilio are required to recruit Brat to HB mRNA and genetic experiments show that Brat is required for repression of HB mRNA (Sonoda, 2001).

Analysis of mutant phenotypes has revealed that Nos and Pum are required for a variety of processes in addition to the development of abdominal segmentation. nos and pum are expressed in tissues other than the female germ line. More important, nos and pum mutants are subviable, revealing an (unknown) essential function for each factor in somatic cells. In the germ line, nos and pum mutants exhibit a number of defects including loss of germ-line stem cells in both sexes, failure of precursor cells to migrate into and populate the somatic gonad, and premature proliferation of precursor cells (pole cells) in the embryo. The premature proliferation appears to result from the inappropriate derepression of maternal Cyclin B (CycB) mRNA in the pole cells; in no other case is the molecular basis of Nos or Pum action currently understood (Sonoda, 2001 and references therein).

It was thus of interest to determine whether Nos and Pum also act in conjunction with Brat to regulate maternal Cyclin B mRNA. Using antibodies directed against different regions of the Brat protein, it has been found that Brat is distributed throughout the syncitial blastoderm stage embryo when HB mRNA is repressed, and is also present in the cytoplasm of the pole cells when maternal Cyclin B mRNA is regulated. However, Cyclin B mRNA is repressed normally in the pole cells of brat^fs mutant embryos, but not in the pole cells of nos mutant embryos. Thus, Brat does not appear to play a role in repression of Cyclin B, although the possibility that the residual activity of Brat^fs1 is sufficient to regulate Cyclin B but not hb cannot be ruled out (Sonoda, 2001).

The cis-acting signals that mediate Nos- and Pum-dependent regulation of Cyclin B have not yet been defined precisely. However, NRE-like sequences are present in the maternal isoform of the Cyclin B mRNA, which is regulated. If indeed Pum, Nos, and NRE-like sequences mediate its regulation, then why would repression of Cyclin B mRNA be Brat independent (Sonoda, 2001)?

To investigate this issue, an examination was made of the binding of Pum, Nos, and Brat to the Cyclin B NRE-like element in vitro. The RNA used in these experiments contains 136 nucleotides that include all of the NRE homologous elements as well as flanking sequences. Pum binds to this Cyclin B-derived RNA in gel mobility-shift experiments, but not to a derivative bearing mutations in the conserved NRE-like element, consistent with the idea that similar sequences in Cyclin B and HB are recognized. Bound Pum can recruit Nos into a ternary complex on Cyclin B RNA, much as it does on the HB NRE. However, the ternary complex assembled on Cyclin B RNA recruits Brat at least 10-fold less efficiently than the corresponding complex assembled on the hb NRE. This surprising observation may in part explain the Brat independence of Cyclin B regulation. Furthermore, it suggests that the RNA sequence specifies the geometry of the Pum/Nos complex, which in turn determines whether Brat is recruited or not (Sonoda, 2001).

Brat acts as a growth suppressor in the larval brain (Arama, 2000). Whether Brat acts by a similar molecular mechanism in the brain and in the early embryo (in regulating HB mRNA) is unclear; however, the observation that single amino acid substitutions in the NHL domain of the Brat^fs mutant proteins disrupt both processes is consistent with such an idea. The role of Brat in the brain is not yet clear, since the phenotype has not been characterized in detail and regulatory targets have yet to be identified (Sonoda, 2001).

The role of Brat in the imaginal discs has been even less clear. Loss of brat function leads to no obvious defects in imaginal development (Arama, 2000), and rare escaper homozygous brat^- flies appear morphologically normal. One role for Brat was revealed by experiments in which imaginal disc tissue was transplanted into the body cavity of adult hosts; brat^- but not wild-type discs metastasize and kill the fly (Woodhouse, 1998). This observation suggests that Brat is expressed in the discs, which led to a consideration of the possibility that loss-of-function mutants exhibit no apparent phenotype due to the presence of a redundant activity (Sonoda, 2001).

To investigate this possibility, either Brat⁺ or Brat^fs1 were misexpressed using an engrailed (en)-GAL4 driver line and UAS transgenes. Flies were examined for phenotypes resulting from the gain of Brat function. Endogenous Brat accumulates uniformly in the cytoplasm of cells in wing discs from third instar larvae. In either UASbrat⁺ or UASbrat^fs1 discs that also bear the enGAL4 driver, a modest excess of protein accumulates in the posterior compartment of the wing disc; analysis of Western blots suggests that the level of overproduction is less than 2-3 fold. At this stage of development, ectopic expression of either protein does not substantially alter the morphology of the discs (Sonoda, 2001).

However, misexpression of Brat⁺ causes an intriguing growth suppression phenotype that is evident in the wings of adults. Three observations stand out: (1) the en;brat⁺ wings are 24% smaller than control wings. They are also usually deformed, probably as a result of poor adhesion between the dorsal and ventral surfaces. (2) The reduction in wing size appears to be due to a reduction in the number of cells contributing to the wing rather than a reduction in the size of the cells. This conclusion is based on a measurement of the density of epidermal hairs, each secreted by a single cell. (3) The phenotype is nonautonomous, extending into the anterior compartment where the en promoter is not active. For example, the anterior-most sector of en;brat⁺ wings (bounded by the first and second longitudinal veins) is on average 22% smaller than the corresponding region of control wings (Sonoda, 2001).

Significantly, none of the phenotypes associated with misexpression of Brat⁺ is caused by misexpression of similar levels of Brat^fs1. This supports the idea that Brat acts by a similar molecular mechanism to regulate abdominal segmentation in the embryo and growth of the wing imaginal disc (Sonoda, 2001).

A model is presented of how Nos, Pum, and Brat act to regulate gene expression. The model involves combinatorial interactions among cis-acting sequences in regulated mRNAs, proteins that recognize these sequences, and the NHL domain of Brat. Recruitment of Brat occurs through protein-protein interactions with RNA-bound Pum and Nos; formation of the resulting quaternary complex is essential for translational control of HB. Recruitment of Brat to the NRE jointly by Nos and Pum is essential for regulation of HB mRNA. Three lines of evidence show that the NHL domain plays a key role in this process: (1) the NHL domain is sufficient to mediate interaction with the Nos/Pum/NRE complex, thereby targeting Brat to HB mRNA; (2) single amino acid substitutions within the NHL domain attenuate interaction with the ternary complex and regulation of hb in vivo; (3) maternal expression of the wild-type NHL domain alone is sufficient to restore HB regulation in brat^fs mutant embryos. This result suggests that the NHL domain contains intrinsic translation regulatory activity. However, activity of the isolated NHL domain is (necessarily) assayed in the presence of Brat^fs mutant protein, and thus, the possibility that the amino-terminal BCC domain participates somehow in HB mRNA regulation cannot be ruled out (Sonoda, 2001).

Brat appears to play no role in regulating Cyclin B mRNA in the pole cells, although Nos and Pum are required for this process. This observation is perhaps not surprising, since translation of Cyclin B mRNA in the posterior region of the syncitial cleavage stage embryo appears to be uninhibited, even in the presence of Nos, Pum, and Brat. Only in the pole cells, which extrude from the posterior extreme of the embryo, is Cyclin B mRNA repressed. Perhaps the specialized pole plasm incorporated into these cells contains a Cyclin B-specific corepressor that acts in conjunction with Nos and Pum. Alternatively, the Nos/Pum complex on Cyclin B mRNA may be sufficient to regulate translation without a cofactor in the pole cells (Sonoda, 2001).

In either case, experiments with Cyclin B reveal an unanticipated complexity: the Nos/Pum complexes assembled on Cyclin B and HB mRNAs apparently have different conformations, as revealed by their ability to interact with Brat. Perhaps the RNA sequence acts as a scaffold, bringing Nos and Pum together on the RNA in different relative orientations in the two cases. Alternatively, the RNA might act as an allosteric effector, altering the conformation of Pum to allow interaction with different cofactors (Sonoda, 2001).

Brat acts as a growth suppressor in the larval brain and, upon modest overexpression, in the wing imaginal disc. Current evidence suggests that, in these tissues, Brat likely acts with cofactors other than Pum or Nos, although the supporting evidence is relatively weak. The brains of mutant larvae bearing the strongest extant alleles of pum do not exhibit a tumorous brat phenotype, consistent with the idea that some other factor acts in conjunction with Brat in this tissue. Attempts were made to test the role of Pum in mediating the en;brat⁺ phenotype, but flies of the appropriate genotype could not be recovered (presumably due to the subviability of both pum^- and en;brat⁺ flies). The role of Nos in mediating Brat action is less easily assessed, since larvae bearing lethal nos alleles die before the third instar when both the brat^- and en;brat⁺ phenotypes are evident. Weaker alleles, such as nos^RC, have substantial residual activity (Sonoda, 2001).

Perhaps the most striking aspect of the en;brat⁺ phenotype is that ectopic Brat suppresses growth nonautonomously. This is in contrast to the action of other growth regulators that have been the focus of recent research in flies. Regulation of cell size and cell number in the imaginal discs is complex. One class of regulators is the extracellular signals of the EGF, TGF-ß, and Wg pathways that coordinately control pattern and growth. Another class consists of signals mediated by molecules such as Ras, Myc, TOR, and members of the insulin receptor pathway that primarily control cell size or number, but not pattern. For many of these 'pure' growth regulators, ectopic expression enhances (or suppresses) growth of the imaginal discs to an extent similar to that observed for Brat. However, in none of these cases is the effect on growth transmitted to surrounding cells, as is true for Brat. Thus, Brat appears to regulate either novel pathways or novel combinations of pathways that generate extracellular signals (Sonoda, 2001).

All three Drosophila NHL proteins regulate some aspect of growth, suggesting this may be a common role for NHL proteins in general. Mutations in mei-P26/CG12218 and dappled/CG1624 reveal that the proteins encoded by these loci suppress growth of melanotic and ovarian tumors, respectively (Rodriguez, 1996; Page, 2000). Mei-P26 is also required for a normal frequency and distribution of genetic exchange during meiosis. Given the structural similarity among the three fly proteins, it was assumed that Dappled and Mei-P26, like Brat, act by regulating translation or some other aspect of mRNA metabolism. Little else is currently known of the cell biological processes controlled by Brat, Dappled, or Mei-P26 (Sonoda, 2001).

Do other NHL proteins act in a manner similar to Brat? Relatively little is known about the molecular mechanism by which these factors act in vivo, and thus it is not clear whether they regulate translation or some other aspect of posttranscriptional gene expression. Nevertheless, an argument for analogous function can be made for three of the family members, based on current knowledge; (1) the HT2A human protein interacts with the site-specific RNA-binding Tat protein (Fridell, 1995), much as Brat interacts with Nos and Pum; (2) C. elegans NCL-1 appears to regulate growth, although the mutant worms have larger cells rather than more cells (Frank, 1998). (3) The most striking analogy with Brat function comes from C. elegans LIN-41, which acts in the penultimate step of the heterochronic pathway (Reinhart, 2000; Slack, 2000). Like Brat, LIN-41 is a posttranscriptional regulator. And like Brat, which acts in concert with Nos and Pum, LIN-41 appears to play a role in the switch from sperm to oocyte production in hermaphrodites that is governed by homologs of Nos and Pum. Thus, it seems likely that LIN-41 and Brat act by a similar mechanism, interacting with RNA-bound factors to repress translation (Sonoda, 2001 and references therein).

GENE STRUCTURE

cDNA clone length - 4933 bases

Bases in 5' UTR - 506

Exons - 5

Bases in 3' UTR - 1313

PROTEIN STRUCTURE

Amino Acids - 1037

Structural Domains

The brat gene was cloned from a transposon-tagged allele and its gene product was identifed. Many NHL (NCL-1, HT2A, and LIN-41) domain proteins, including Brat, share motifs: a Ring-finger, one or two B-box motifs, and a coiled-coil (RBCC) (Slack, 1998). brat encodes for a 1037 amino acid protein with an N-terminal B-boxl zinc finger followed by a B-box2 zinc finger, a coiled-coil domain, and a C-terminal beta-propeller domain with six blades. All these motifs are known to mediate protein-protein interactions. Two other brat-like genes have been identified in Drosophila (mei-P26/CG12218 and dappled/CG1624), and homologs were identified in the nematode, mouse, rat, and human (Arama, 2000).

date revised: 22 April 2001

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