Robin P. Wharton

Binding of pumilio to maternal hunchback mRNA is required for posterior patterning in Drosophila embryos.

Posterior patterning in Drosophila embryos is governed by nanos (nos), which acts by repressing the translation of maternal transcripts of the hunchback (hb) gene. Sites in hb mRNA that mediate this repression, named nanos response elements (NREs), have been identified. However, we know of no evidence of a direct interaction between nos, or any other protein, and the NRE. Here, we show that two proteins present in embryonic extracts, neither one nos, bind specifically to the NRE in vitro. Furthermore, we show that binding in vitro correlates with NRE function in vivo. One of the NRE-binding factors is encoded by pumilio (pum), a gene that, like nos, is essential for abdominal segmentation. These and other observations suggest that pum acts by recognizing the NRE and then recruiting nos. Presumably, the resulting complex inhibits some component of the translation machinery.

Smaug, a Novel RNA-Binding Protein that Operates a Translational Switch in Drosophila

During Drosophila embryogenesis, a gradient of Nanos protein emanating from the posterior pole organizes abdominal segmentation. This gradient arises from translational regulation of nanos mRNA, which is activated in the specialized cytoplasm at the posterior pole of the embryo and repressed elsewhere. Previously, we have defined cis-acting elements in the mRNA that mediate this translational switch. In this report, we identify a factor named Smaug that binds to these elements and represses translation in the bulk cytoplasm. Smaug interacts gentically and biochemically with Oskar, a key component of the pole plasm for activation of nanos mRNA and specification of the germline precursors. These observations suggest that Smaug operates a translational switch that governs the distribution of Nanos protein.

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.

E2F mediates developmental and cell cycle regulation of ORC1 in Drosophila

Throughout the cell cycle of Saccharomyces cerevisiae, the level of origin recognition complex (ORC) is constant and ORCs are bound constitutively to replication origins. Replication is regulated by the recruitment of additional factors such as CDC6. ORC components are widely conserved, and it generally has been assumed that they are also stable factors bound to origins throughout the cell cycle. In this report, we show that the level of the ORC1 subunit changes dramatically throughout Drosophila development. The accumulation of ORC1 is regulated by E2F‐dependent transcription. In embryos, ORC1 accumulates preferentially in proliferating cells. In the eye imaginal disc, ORC1 accumulation is cell cycle regulated, with high levels in late G1 and S phase. In the ovary, the sub‐nuclear distribution of ORC1 shifts during a developmentally regulated switch from endoreplication of the entire genome to amplification of the chorion gene clusters. Furthermore, we find that overexpression of ORC1 alters the pattern of DNA synthesis in the eye disc and the ovary. Thus, replication origin activity appears to be governed in part by the level of ORC1 in Drosophila.

RNA Recognition via the SAM Domain of Smaug

The Nanos protein gradient in Drosophila, required for proper abdominal segmentation, is generated in part via translational repression of its mRNA by Smaug. We report here the crystal structure of the Smaug RNA binding domain, which shows no sequence homology to any previously characterized RNA binding motif. The structure reveals an unusual makeup in which a SAM domain, a common protein-protein interaction module, is affixed to a pseudo-HEAT repeat analogous topology (PHAT) domain. Unexpectedly, we find through a combination of structural and genetic analysis that it is primarily the SAM domain that interacts specifically with the appropriate nanos mRNA regulatory sequence. Therefore, in addition to their previously characterized roles in protein-protein interactions, some SAM domains play crucial roles in RNA binding.

mRNA Regulation by Puf Domain Proteins

Puf domain proteins bind specific sequences in mRNAs to regulate their translation or stability, or both. Neither the mechanism of their action nor the identities of targeted mRNAs have been well defined. Recent work suggests that Puf proteins generally act by recruiting Pop2, a deadenylation enzyme that is part of a large complex. Recent work from a separate group defines a subset of the Drosophila transcriptome that is bound by the fly Puf protein, Pumilio. Together, these papers substantially increase our understanding of the biology of the Puf family of mRNA regulators.

The molecular chaperone Hsp90 is required for mRNA localization in Drosophila melanogaster embryos

Localization of maternal nanos mRNA to the posterior pole is essential for development of both the abdominal segments and primordial germ cells in the Drosophila embryo. Unlike maternal mRNAs such as bicoid and oskar that are localized by directed transport along microtubules, nanos is thought to be trapped as it swirls past the posterior pole during cytoplasmic streaming. Anchoring of nanos depends on integrity of the actin cytoskeleton and the pole plasm; other factors involved specifically in its localization have not been described to date. Here we use genetic approaches to show that the Hsp90 chaperone (encoded by Hsp83 in Drosophila) is a localization factor for two mRNAs, nanos and pgc. Other components of the pole plasm are localized normally when Hsp90 function is partially compromised, suggesting a specific role for the chaperone in localization of nanos and pgc mRNAs. Although the mechanism by which Hsp90 acts is unclear, we find that levels of the LKB1 kinase are reduced in Hsp83 mutant egg chambers and that localization of pgc (but not nos) is rescued upon overexpression of LKB1 in such mutants. These observations suggest that LKB1 is a primary Hsp90 target for pgc localization and that other Hsp90 partners mediate localization of nos.

Translational repressors in Drosophila

Translational regulation is an important aspect of gene regulation, particularly during early development of the fruit fly embryo when transcriptional mechanisms are untenable. Study of pattern formation and dosage compensation has identified several repressors that bind discrete sites in the untranslated portions of target mRNAs. These repressors do not work in isolation - each binds multiple sites in the appropriate mRNA, and the resulting RNA-protein complexes appear to recruit co-repressors by a variety of mechanisms.

E(nos)/CG4699 required for nanos function in the female germ line of Drosophila

The translational repressor Nanos is required in the germ line stem cells of the Drosophila ovary to maintain their capacity for self‐renewal. Following division of the stem cells, Nanos is inhibited in the daughters that differentiate into cysts and ultimately become mature oocytes. The control of Nanos activity is thus an important aspect of the switch from self‐renewal to differentiation. In this report, we describe a genetic interaction between nanos and Enhancer of nos, an allele of the previously uncharacterized locus CG4699. We find that E(nos) protein is required for normal accumulation of Nanos in the ovary and thus for maintenance of the germ line. The mechanism by which E(nos)/CG4699 protein acts is not clear, although it has been found in a complex with Mof acetylase. Consistent with the finding that E(nos) interacts with Mof, we observe that nanos and mof also interact genetically to maintain normal oogenesis. genesis 48:161–170, 2010.

A splicer that represses (translation)

Regulated translation and subcellular localization of maternal mRNAs underlies establishment of the antero-posterior axis in the Drosophila oocyte. In this issue of Genes & Development, Besse et al. (pp. 195-207) show that a molecule better known as a regulator of alternative splicing in the nucleus, polypyrimidine tract-binding protein (PTB), is required for repression of oskar mRNA in the cytoplasm. Their work suggests that PTB need not engage oskar mRNA in the nucleus for efficient repression, providing an important counterexample to the increasingly popular idea that cytoplasmic regulation initiates in the nucleus.