Drosophila Pumilio protein contains multiple autonomous repression domains that regulate mRNAs independently of Nanos and brain tumor

Drosophila melanogaster Pumilio is an RNA-binding protein that potently represses specific mRNAs. In developing embryos, Pumilio regulates a key morphogen, Hunchback, in collaboration with the cofactor Nanos. To investigate repression by Pumilio and Nanos, we created cell-based assays and found that Pumilio inhibits translation and enhances mRNA decay independent of Nanos. Nanos robustly stimulates repression through interactions with the Pumilio RNA-binding domain. We programmed Pumilio to recognize a new binding site, which garners repression of new target mRNAs. We show that cofactors Brain Tumor and eIF4E Homologous Protein are not obligatory for Pumilio and Nanos activity. The conserved RNA-binding domain of Pumilio was thought to be sufficient for its function. Instead, we demonstrate that three unique domains in the N terminus of Pumilio possess the major repressive activity and can function autonomously. The N termini of insect and vertebrate Pumilio and Fem-3 binding factors (PUFs) are related, and we show that corresponding regions of human PUM1 and PUM2 have repressive activity. Other PUF proteins lack these repression domains. Our findings suggest that PUF proteins have evolved new regulatory functions through protein sequences appended to their conserved PUF repeat RNA-binding domains.     

Nanos and Pumilio are essential for dendrite morphogenesis in Drosophila peripheral neurons

Much attention has focused on dendritic translational regulation of neuronal signaling and plasticity. For example, long-term memory in adult Drosophila requires Pumilio (Pum), an RNA binding protein that interacts with the RNA binding protein Nanos (Nos) to form a localized translation repression complex essential for anterior-posterior body patterning in early embryogenesis. Whether dendrite morphogenesis requires similar translational regulation is unknown. Here we report that nos and pum control the elaboration of high-order dendritic branches of class III and IV, but not class I and II, dendritic arborization (da) neurons. Analogous to their function in body patterning, nos and pum require each other to control dendrite morphogenesis, a process likely to involve translational regulation of nos itself. The control of dendrite morphogenesis by Nos/Pum, however, does not require hunchback, which is essential for body patterning. Interestingly, Nos protein is localized to RNA granules in the dendrites of da neurons, raising the possibility that the Nos/Pum translation repression complex operates in dendrites. This work serves as an entry point for future studies of dendritic translational control of dendrite morphogenesis.     

Translational repression restricts expression of the C. elegans Nanos homolog NOS-2 to the embryonic germline

Members of the nanos gene family are evolutionarily conserved regulators of germ cell development. In several organisms, Nanos protein expression is restricted to the primordial germ cells (PGCs) during early embryogenesis. Here, we investigate the regulation of the Caenorhabditis elegans nanos homolog nos-2. We find that the nos-2 RNA is translationally repressed. In the adult germline, translation of the nos-2 RNA is inhibited in growing oocytes, and this inhibition depends on a short stem loop in the nos-2 3'UTR. In embryos, nos-2 translation is repressed in early blastomeres, and this inhibition depends on a second region in the nos-2 3'UTR. nos-2 RNA is also degraded in somatic blastomeres by a process that is independent of translational repression and requires the CCCH finger proteins MEX-5 and MEX-6. Finally, the germ plasm component POS-1 activates nos-2 translation in the PGCs. A combination of translational repression, RNA degradation, and activation by germ plasm has also been implicated in the regulation of nanos homologs in Drosophila and zebrafish, suggesting the existence of conserved mechanisms to restrict Nanos expression to the germline.     

Maternal Pumilio acts together with Nanos in germline development in Drosophila embryos

The maternal RNA-binding proteins Pumilio (Pum) and Nanos (Nos) act together to specify the abdomen in Drosophila embryos. Both proteins later accumulate in pole cells, the germline progenitors. Nos is required for pole cells to differentiate into functional germline. Here we show that Pum is also essential for germline development in embryos. First, a mutation in pum causes a defect in pole-cell migration into the gonads. Second, in such pole cells, the expression of a germline-specific marker (PZ198) is initiated prematurely. Finally, pum mutation causes premature mitosis in the migrating pole cells. We show that Pum inhibits pole-cell division by repressing translation of cyclin B messenger RNA. As these phenotypes are indistinguishable from those produced by nos mutation, we conclude that Pum acts together with Nos to regulate these germline-specific events.     

Translational repression: not just a Puf of smoke

Proteins containing Puf domains interact with cofactors to form complexes that bind RNAs and control diverse developmental events. Recent studies have shed light on how the Puf family of proteins regulates mRNA activity.     

Maternal Nanos and Pumilio regulate zygotic vasa expression autonomously in the germ-line progenitors of Drosophila melanogaster embryos

vasa (vas) is transcribed earliest among reported genes expressed in the germ-line progenitors, or pole cells, in Drosophila melanogaster embryos. Its expression is detected in the germ-line cells throughout their development, making vas expression a useful marker for the establishment of germ-line fate. In the present report, it is shown that maternal Nos and Pum are required for normal expression of vas in pole cells. First, expression of enhancer-trap marker BC69, which reflects vas expression, is promoted by maternal Nos and Pum. Second, expression of vas mRNA in pole cells is promoted by maternal Nos and Pum. Third, pole cell transplantation experiments reveal that maternal Nos and Pum are required autonomously in pole cells for proper expression of vas. Finally, Nos and Pum are dispensable for vas expression in oogenesis, although they are expressed zygotically in adult ovaries. These observations show that germ-line-specific vas expression is promoted by autonomous function of maternal Nos and Pum in the germ-line progenitors during embryogenesis, and is regulated differentially in embryogenesis and oogenesis.     

Translational control of maternal Cyclin B mRNA by Nanos in the Drosophila germline

In the Drosophila embryo, Nanos and Pumilio collaborate to repress the translation of hunchback mRNA in the somatic cytoplasm. Both proteins are also required for repression of maternal Cyclin B mRNA in the germline; it has not been clear whether they act directly on Cyclin B mRNA, and if so, whether regulation in the presumptive somatic and germline cytoplasm proceeds by similar or fundamentally different mechanisms. In this report, we show that Pumilio and Nanos bind to an element in the 3' UTR to repress Cyclin B mRNA. Regulation of Cyclin B and hunchback differ in two significant respects. First, Pumilio is dispensable for repression of Cyclin B (but not hunchback) if Nanos is tethered via an exogenous RNA-binding domain. Nanos probably acts, at least in part, by recruiting the CCR4-Pop2-NOT deadenylase complex, interacting directly with the NOT4 subunit. Second, although Nanos is the sole spatially limiting factor for regulation of hunchback, regulation of Cyclin B requires another Oskar-dependent factor in addition to Nanos. Ectopic repression of Cyclin B in the presumptive somatic cytoplasm causes lethal nuclear division defects. We suggest that a requirement for two spatially restricted factors is a mechanism for ensuring that Cyclin B regulation is strictly limited to the germline.     

Translational repression as a conserved mechanism for the regulation of embryonic polarity

The mechanisms used to establish embryonic polarity are still largely unknown. A recent paper⁽¹⁾ describes the expression pattern of the gene glp-1, which is required for induction events during development of the nematode Caenorhabditis elegans. Although glp-1 RNA is found throughout the early embryo, Glp-1 protein is only expressed in anterior cells. This negative translational regulation in posterior cells is shown to be mediated through sequences in the glp-1 3′ untranslated region (3′UTR). Thus in nematodes, as in Drosophila, translational repression is one mechanism used to establish the embryonic anterior-posterior axis.     

Biochemical identification of Xenopus Pumilio as a sequence-specific cyclin B1 mRNA-binding protein that physically interacts with a Nanos homolog, Xcat-2, and a cytoplasmic polyadenylation element-binding protein

Translational activation of dormant cyclin B1 mRNA stored in oocytes is a prerequisite for the initiation or promotion of oocyte maturation in many vertebrates. Using a monoclonal antibody against the domain highly homologous to that of Drosophila Pumilio, we have shown for the first time in any vertebrate that a homolog of Pumilio is expressed in Xenopus oocytes. This 137-kDa protein binds to the region including the sequence UGUA at nucleotides 1335-1338 in the 3'-untranslated region of cyclin B1 mRNA, which is close to but does not overlap the cytoplasmic polyadenylation elements (CPEs). Physical in vitro association of Xenopus Pumilio with a Xenopus homolog of Nanos (Xcat-2) was demonstrated by a protein pull-down assay. The results of immunoprecipitation experiments showed in vivo interaction between Xenopus Pumilio and CPE-binding protein (CPEB), a key regulator of translational repression and activation of mRNAs stored in oocytes. This evidence provides a new insight into the mechanism of translational regulation through the 3'-end of mRNA during oocyte maturation. These results also suggest the generality of the function of Pumilio as a translational regulator of dormant mRNAs in both invertebrates and vertebrates.     

Translational repression: biological activity of plasmid-encoded bacteriophage T4 RegA protein

The RegA protein of bacteriophage T4 is a translational repressor that regulates expression of several phage early mRNAs. We have cloned wild-type and mutant alleles of the T4 regA gene under control of the heat-inducible, plasmid-borne leftward promoter (PL) of phage lambda. Expression of the cloned regA+ gene resulted in the synthesis of a protein that closely resembled phage-encoded RegA protein in biological properties. It repressed its own synthesis (autogenous translational control) as well as the synthesis of specific T4-encoded proteins that are known from other studies to be under RegA-mediated translational control. Cloned mutant alleles of regA exhibited derepressed synthesis of the mutant regA gene products and were ineffective in trans against RegA-sensitive mRNA targets. The effects of plasmid-encoded RegA proteins were also demonstrated in experiments using two compatible plasmids in uninfected Escherichia coli. The two-plasmid assays confirm the sensitivities of several cloned T4 genes to RegA-mediated translational repression and are well-suited for genetic analysis of RegA target sites. Repression specificity in this system was demonstrated by using wild-type and operator-constitutive translational initiation sites of T4 rIIB fused to lacZ. The results show that no additional T4 products are required for RegA-mediated translational repression. Additional evidence is provided for the proposal that uridine-rich mRNA sequences are preferred targets for the repressor. Surprisingly, plasmid-generated RegA protein represses the synthesis of some E. coli proteins and appears to enhance selectively the synthesis of others. The RegA protein may have multiple functions, and its binding sites are not restricted to phage mRNAs.     

Biochemical characterization of Pumilio1 and Pumilio2 in Xenopus oocytes

Precise control of the timing of translational activation of dormant mRNAs stored in oocytes is required for normal progression of oocyte maturation. We previously showed that Pumilio1 (Pum1) is specifically involved in the translational control of cyclin B1 mRNA during Xenopus oocyte maturation, in cooperation with cytoplasmic polyadenylation element-binding protein (CPEB). It was reported that another Pumilio, Pumilio2 (Pum2), exists in Xenopus oocytes and that this protein regulates the translation of RINGO mRNA, together with Deleted in Azoospermia-like protein (DAZL). In this study, we characterized Pum1 and Pum2 biochemically by using newly produced antibodies that discriminate between them. Pum1 and Pum2 are bound to several key proteins involved in translational control of dormant mRNAs, including CPEB and DAZL, in immature oocytes. However, Pum1 and Pum2 themselves have no physical interaction. Injection of anti-Pum1 or anti-Pum2 antibody accelerated CPEB phosphorylation, cyclin B1 translation, and oocyte maturation. Pum1 phosphorylation coincides with the dissociation of CPEB from Pum1 and the translational activation of cyclin B1 mRNA, a target of Pum1, whereas Pum2 phosphorylation occurred at timing earlier than that for Pum1. Some, but not all, of cyclin B1 mRNAs release the deadenylase PARN during oocyte maturation, whereas Pum1 remains associated with the mRNA. On the basis of these findings, we discuss the functions of Pum1 and Pum2 in translational control of mRNAs during oocyte maturation.     

Functional redundancy among Nanos proteins and a distinct role of Nanos2 during male germ cell development

The mouse Nanos proteins, Nanos2 and Nanos3, are required for germ cell development and share a highly conserved zinc-finger domain. The expression patterns of these factors during development, however, differ from each other. Nanos3 expression in the mouse embryo commences in the primordial germ cells (PGCs) just after their formation, and a loss of this protein results in the germ cell-less phenotype in both sexes. By contrast, Nanos2 expression begins only in male PGCs after their entry into the genital ridge and a loss of this protein results in a male germ cell deficiency, irrespective of the co-expression of Nanos3 in these cells. These results indicate that these two Nanos proteins have distinct functions, which depend on the time and place of their expression. To further elucidate this, we have generated transgenic mouse lines that express Nanos2 under the control of the Oct4DeltaPE promoter and examined Nanos2 function in a Nanos3-null genetic background. We find that ectopically produced Nanos2 protein rescues the Nanos3-null defects, because the germ cells fully develop in both sexes in the transgenic mice. This result indicates that Nanos2 can substitute for Nanos3 during early PGC development. By contrast, our current data show that Nanos3 does not rescue the defects in Nanos2-null mice. Our present findings thus indicate that there are redundant functions of the Nanos proteins in early PGC development, but that Nanos2 has a distinct function during male germ cell development in the mouse.     

Crystal structure of a Pumilio homology domain

Puf proteins regulate translation and mRNA stability by binding sequences in their target RNAs through the Pumilio homology domain (PUM-HD), which is characterized by eight tandem copies of a 36 amino acid motif, the PUM repeat. We have solved the structure of the PUM-HD from human Pumilio1 at 1.9 A resolution. The structure reveals that the eight PUM repeats correspond to eight copies of a single, repeated structural motif. The PUM repeats pack together to form a right-handed superhelix that approximates a half doughnut. The distribution of side chains on the inner and outer faces of this half doughnut suggests that the inner face of the PUM-HD binds RNA while the outer face interacts with proteins such as Nanos, Brain Tumor, and cytoplasmic polyadenylation element binding protein.