The pumilio is a maternal effect gene required for development of the fruit fly Drosophila melanogaster. pumilio is required during oogenesis for the development and maintenance of the germ-line stem cell lineage and in the embryo for patterning of the abdomen. pumilio, in concert with nanos, represses translation of maternally provided hunchback messenger RNA in the Drosophila embryo. Translational regulation is mediated by sequences in the 3′ untranslated region of hunchback, and Pumilio binds specifically to these regulatory sequences. Pumilio is a member of a conserved family of RNA-binding proteins with a novel RNA-binding motif. Ongoing studies seek to understand the interaction of the Pumilio protein with its RNA target.
0.1. Protein and RNA Structure
The pumilio gene spans over 160 kbp. The primary transcript contains 12 introns (one over 120 kbp) and produces two mature mRNAs (6.8 kb and 6.7 kb) that encode identical proteins, but use different first exons. The pumilio mRNA is detected at high levels in ovaries, 0 to 8 hour embryos, and adult females (1, 2). Low levels of the transcript are also detected in 8- to 24-h embryos and pupae. The Pumilio protein is a 1533-amino-acid-residue polypeptide with a predicted molecular weight of 160 kDa. Analysis of Pumilio protein reveals eight imperfect repeats of 36 residues each (1). Recently it has been shown that these repeats constitute the pumilio RNA-binding domain (3, 4). This RNA-binding motif (known as the pumilio homology domain or PUM-HD) is conserved from yeast to humans (3, 5).
1. pumilio in Oogenesis
A Drosophila melanogaster ovary consists of approximately 16 ovarioles each representing an independent egg assembly line (for review see Ref. 6). Every ovariole is a linear array of developing egg chambers, beginning at the anterior with the germarium, where the progeny of the germ-line and somatic stem cells are organized into egg chambers. At the tip of the germarium, the germ-line stem cell produces a new stem cell and a cystoblast by asymmetric division. The cystoblast undergoes four rounds of division with incomplete cytokinesis, resulting in 16 cells connected by cytoplasmic bridges; one of these cells becomes the oocyte while the other 15 become nurse cells. Somatically derived follicle cells surround the egg chamber. As the egg chambers mature, the nurse cells synthesize and deposit factors into the oocyte that are required for the development of the oocyte and early embryo. Late in oogenesis, the nurse cells contract and dump the contents of their cytoplasm into the oocyte.
Pumilio protein is expressed at high levels in the germ line stem cells and at lower levels in the dividing cystoblasts and the soma (7, 8). Females trans-heterozygous for strongpumilio alleles lay a few eggs, but rapidly become sterile due to a loss of germ-line stem cells from the ovary (7, 8). Analysis of pumilio mutant ovaries demonstrates that germ cells often fail to be incorporated into the ovarioles, suggesting that pumilio, like nanos, is required for germ-line cells to populate the ovary. Ovarioles with germ-line cells lack germ-line stem cells, and germline cells differentiate directly into egg chambers. This suggests that pumilio is required for establishment of the germ-line stem cell fate. Additionally, pumilio mutant ovaries from older flies often contain tumorous growths resulting from overproliferation of somatic cells, suggesting a role for pumilio in the soma as well.
2. pumilio in Embryonic Patterning
In the late oocyte and early embryo, pumilio mRNA and protein are distributed throughout the embryo (2). Pumilio activity is required for the translational repression of maternal hunchback RNA in the posterior half of the embryo. Hunchback is a zinc-finger transcription factor that controls transcription of the abdominal gap genes (9). In addition to Pumilio, repression of maternal hunchback translation is mediated by sequences within the hunchback 3′-untranslated region (known as nanos response elements or NREs) and the activity of the posterior determinant nanos (10). Pumilio protein binds to the hunchback NREs (3, 4, 11). Mutations that disrupt Pumilio binding in vitro have a strong effect on the regulation of hunchback in vivo (4). Together Pumilio and Nanos act through the hunchback NREs to direct removal of the maternal hunchback poly (A) tail in the posterior of the embryo, suggesting that deadenylation mediates translational repression (12). Ectopic expression of Nanos in the eye imaginal disc results in disruption of ommatidial development (4). Loss of Pumilio function suppresses the activity of ectopic Nanos, suggesting that nanos and pumilio are cooperating to regulate some gene product required for eye development. Additionally, ectopic nanos can repress the expression of reporter genes bearing the hunchback NREs and an internal ribosome entry site, suggesting that nanos and pumilio regulate translation independent of the 5′-cap (4).
3. The Pumilio Homology RNA-Binding Domain
The pumilio homology domain has been identified in proteins or expressed sequence tags from human, mouse (M. musculus), rat (R. norvegicus ), nematodes (C. elegans ), yeast (S. cerevisia and S. pombe), plants (A. thaliana), rice (O. sativa), and corn ( Z. mays) (3, 5). The high degree of sequence conservation among these very diverse species suggests that the PUM-HD is an ancient protein motif. Furthermore, a human PUM-HD can bind to the hunchback NREs, albeit with slightly altered sequence specificity (3). fem-3 is a gene required to determine the sexual fate of C. elegans hermaphrodite germ line (13, 14). The regulation offem-3 is very analogous to that seen for Drosophila hunchback. fem-3 contains a small cis-element within its 3′-untranslated region (known as the point-mutation element) that, when mutated, causes aberrant translation offem-3 (15, 16). Recently, FBF-1 and FBF-2 were cloned based on their ability to bind to the wild type, but not mutant, point-mutation element. FBF-1 and FBF-2 each contain a PUM-HD, suggesting that these proteins share not only a conserved protein motif with pumilio, but also a conserved function (5). Other PUM-HD family members have recently been shown to have a role in aging and recovery from stress, but their mechanism of action remains unclear (17).
