BIOLOGICAL OVERVIEW

pumilio is a posterior group gene: genes in this group are considered essential for the development of the posterior of the fly. The protein is supplied by the mother, and is ready to go to work even before fertilization takes place. A discussion of the PUM protein is clarified by looking at what takes place just before the protein functions. For PUM, this requires an examination of Nanos. Nanos governs the posterior patterning of Drosophila embryos. Localization of NOS mRNA assures that NOS protein is present in the posterior and can thus carry out inhibition of translation of Hunchback mRNA, thus allowing for the development of posterior fate.

Experiments have suggested that NOS mRNA is properly localized in pum- mutants, indicating that pum must function after the localization of NOS mRNA (Barker, 1992). It has been assumed that NOS itself is sufficient for inhibition of hunchback translation. The recent observation that PUM protein binds specifically to the Nanos response elements (NRE) of HB mRNA now clarifies the roles of NOS and PUM proteins in repression of HB mRNA translation. PUM protein is recruited first to HB mRNA, followed presumbly by NOS, recruited via protein-protein interactions. The two proteins together act as translational inhibitors (Murata, 1995).

Pumilio has an earlier role in regulation of asymmetric division of germline stem cells in the Drosophila ovary. pumilio mutations are known to affect the asymmetric division of germline stem cells in the Drosophila ovary. Germline stem cells play a pivotal role in gametogenesis; yet little is known about how they are formed, how they divide to self-renew, and how these processes are genetically controlled. Self-renewing asymmetric division of germline stem cells takes place in the Drosophila ovarian germline, as marked by the spectrosome, a cytoplasmic structure rich in membrane skeletal proteins including Spectrin. In the Drosophila ovary, germline stem cells, whose progeny ultimately give rise to eggs, are among the 3 to 5 most apically located germ cells in the germarium -- they are located towards the narrow end of the ovary, at the opposite end from the mature eggs. Terminal filament cells, somatic cells that make up the most proximal part of the ovary close to the stem cells, stain strongly for anti-alpha spectin antibody. The base of the terminal filament contains two to three squamous somatic cells rather than a single basal cell. The basal terminal filament cells retain strong anti-spectrin staining. These basal cells are in contact with two to three underlying germ cells. In these germ cells the spectrosome is usually apically located in the cytoplasm, closely apposed to the basal terminal filament cells. Occasionally, the spectrosome is not in contact with the basal cells. Even so, it is still tethered to the basal cell by a thin filament (Lin, 1997).

The ontogeny of the spectrosome marks the lineage of germline stem cells. The 2-3 most apical germ cells contacting the basal terminal filament cells show striking asymmetry in two ways with regard to the behavior of the spectrosome during mitosis: (1) one pole of their mitotic spindles is always associated with the spectrosome and the terminal filament, clearly marking a cytological asymmetry of the division. (2) The mitotic spindles are oriented along the apico-basal axis of the germarium generating a daughter cell in contact with the basal cell and another daughter cell that is one cell away from the basal cell. This distal daughter cell undergoes incomplete divisions to form germline cysts that ultimately give rise to nurse cells and the egg. Mutations have been identified in which the divisional asymmetry is disrupted. One set of mutations, referred to as ovarette (ovt) mutations, corresponds to a novel class of mutations in the pumilio locus (Lin, 1997).

pumilio mutation produces a small ovary phenotype. To examine whether this phenotype is the result of stem cell dysfunction, the germaria of females bearing a strong ovt mutation were examined. None of the mutant germ cells in pum mutant germaria appear to undergo asymmetric divisions. Instead, they contain only two or three clusters of apparently undifferentiated germline cells that do not contain a spectrosome. This defect suggests that the 2-3 mutant germline stem cells have undergone symmetric divisions to produce clusters of undifferentiated germline cells. Interestingly, lack of a spectrosome does not need to affect the rate of stem cell division. Mutation in the gene hu li tai shao (hts) coding for the cytoskeletal protein adducin, abolishes the spectrosome but does not affect the rate of stem cell division. In hts mutant females lacking a spectrosome, cysts continue to be produced at essentially normal rates. However, hts cystoblasts undergo a drastically modified process of cyst formation and are unable to differentiate an oocyte, presumably due to their inability to support a spectrosome derived fusome (Lin, 1997).

Since pumilio is known to posttranscriptionally repress the expression of Nanos at the earliest stages of germ cell development (Kobayashi, 1996), these results suggest that a similar activity is needed to maintain germ line stem cells. The simplest interpretation of the role of Pumilio in stem cell dynamics is that Pum, and likely Nos as well, act in the stem cell itself where they might act in concert with germ cell-specific molecules, such as Vasa, to participate in translational suppression, which keeps certain genes inactive until specific times in development or in the cell cycle. Inappropriate expression of the suppressed genes would cause the stem cells to assume a cystoblast identity or to proceed down an abnormal developmental pathway leading to the undifferentiated cell clusters (Lin, 1997).

Besides playing a role in ovaries in germ cell development, Pum also acts during embryogenesis to regulate germline development. The maternal RNA-binding proteins Pumilio (Pum) and Nanos (Nos) accumulate in pole cells, the germline progenitors. Nos is required for pole cells to differentiate into functional germline. Pum is also essential for germline development in embryos. A mutation in pum causes a defect in pole-cell migration into the gonads. In such pole cells, the expression of a germline-specific marker (PZ198) is initiated prematurely. pum mutation causes premature mitosis in the migrating pole cells. Pum inhibits pole-cell division by repressing translation of cyclin B messenger RNA. Because these phenotypes are indistinguishable from those produced by nos mutation, it is concluded that Pum acts together with Nos to regulate these germline-specific events (Asaoka-Taguchi, 1999).

Pole cells formed in embryos lacking Pum (pum embryos) were transplanted into wild-type host embryos. The transplanted pum pole cells pass normally through the midgut epithelium into the haemocoel. However, none of the transplanted pum pole cells are incorporated within the gonads of the hosts, whereas normal pole cells taken from control embryos were observed in the gonads. All of the transplanted pum pole cells remain in the haemocoel and the gut lumen. These results show that Pum is autonomously required in pole cells for their migration into the gonads. The expression of the enhancer-trap marker PZ198 was studied in pum pole cells. PZ198 expression, which is normally initiated in pole cells within the gonads, begins prematurely during pole-cell migration in embryos lacking Nos (nos embryos). Similarly, PZ198 expression begins prematurely, at stage 7, in pum mutant pole cells, as compared with stage 13 in control embryos. Thus Pum is also required to repress the premature expression of the enhancer-trap marker in pole cells. The effects of pum and nos mutations on cell-cycle arrest were studied during pole-cell migration. In normal development, pole cells remain quiescent in G2 phase of the cell cycle during stages 7-15. It is expected that Nos and Pum repress the entry of pole cells into mitosis, because pole cells initiate cell division just after Nos becomes undetectable in pole cells at stage 15. To monitor the cell cycle in pole cells, antibodies against a phosphorylated form of histone H3 (PH3) and cyclin E were used. PH3 is detectable in mitosis but is absent during interphase, whereas cyclin E is expressed specifically in S and G2 phases. The disappearance of cyclin E from pole cells is linked to cell-cycle progression from G2 to G1 phase, whereas cyclin E is not degraded during cell cycling of somatic cells. Consistent with the observation that migrating pole cells in wild-type embryos are arrested in G2 phase, almost all pole cells in stage 7-15 embryos show cyclin E staining, but not PH3 staining. In contrast, in pum and nos embryos, the percentage of pole cells expressing cyclin E gradually decreases during stages 7-15, and PH3-positive pole cells became detectable during these stages. Thus, the mutant pole cells are prematurely released from G2 arrest and enter into mitosis. Taken together, these observations show that Pum and Nos are both required for the repression of the G2/M transition in the migrating pole cells (Asaoka-Taguchi, 1999).

Since pum and nos mutations do not affect posterior localization of maternal cyclin B mRNA or its partitioning into pole cells, it is concluded that translation of Cyclin B mRNA is usually repressed by Pum and Nos in pole cells. Cyclin B mRNA contains an NRE-like sequence in its 3' UTR, called the translation-control element (TCE). Deletion of the TCE from the 3' UTR of an epitope-tagged Cyclin B mRNA results in a phenotype similar to that caused by nos and pum mutations. These observations lead to the conclusion that Pum/Nos-dependent translational repression of cyclin B mRNA is mediated by the TCE. Given that Pum binds to the NRE in vitro, it is reasonable to suggest that Pum binds directly to the TCE. This is the first demonstration that maternal factors regulate the translation of specific mRNA in germline progenitors (Asaoka-Taguchi, 1999).

GENE STRUCTURE

Bases in 5' UTR -883 and 1044

Exons - 12

Bases in 3' UTR - 2173

PROTEIN STRUCTURE

Amino Acids - 1533

Structural Domains

The pum gene is unusually large; comparison of genomic and cDNA sequences reveals that the pum transcription unit is at least 160 kb in length. The pum cDNA encodes a 157 x 10(3) M(r) protein which consists mainly of regions enriched in single amino acid repeats, usually glycine, alanine, glutamine or serine/threonine. Six tandem repeats of a 36 amino acid repeat unit are also present (Macdonald, 1992).

Pumilio is the prototypical member of an RNA-binding protein family evolutionarily conserved from yeast to humans. Its signature domain is termed a Puf motif after Drosophila Pumilio and the C. elegans translational regulator FBF (fem-3-binding factor). Puf proteins are implicated in post-transcriptional gene expression in S. cerevisiae, C. elegans, X. laevis and Drosophila. In most characterized situations, these proteins function with Nanos or Nanos-like partners (Gamberi, 2002 and references therein).

EVOLUTIONARY HOMOLOGS

Eukaryotic post-transcriptional regulation is often specified by control elements within mRNA 3'- untranslated regions (3'-UTRs). In order to identify proteins that regulate specific mRNA decay rates in Saccharomyces cerevisae, the role of five members of the Puf family present in the yeast genome (referred to as JSN1/PUF1, PUF2, PUF3, PUF4 and MPT5/PUF5) was analyzed. Yeast strains lacking all five Puf proteins show differential expression of numerous yeast mRNAs. Examination of COX17 mRNA indicates that Puf3p specifically promotes decay of this mRNA by enhancing the rate of deadenylation and subsequent turnover. Puf3p also binds to the COX17 mRNA 3'-UTR in vitro. This indicates that the function of Puf proteins as specific regulators of mRNA deadenylation has been conserved throughout eukaryotes. In contrast to the case in Caenorhabditis elegans and Drosophila, yeast Puf3p does not affect translation of COX17 mRNA. These observations indicate that Puf proteins are likely to play a role in the control of transcript-specific rates of degradation in yeast by interacting directly with the mRNA turnover machinery (Olivas, 2000).

Drosophila Pumilio (Pum) and C. elegans FBF bind to the 3'-untranslated region (3'-UTR) of their target mRNAs and repress translation. Pum and FBF are members of a large and evolutionarily conserved protein family, the Puf family, found in Drosophila, C.elegans, humans and yeasts. Budding yeast, Saccharomyces cerevisiae, has five proteins with conserved Puf motifs: Mpt5/Uth4, Ygl014w, Yll013c, Jsn1 and Ypr042c. Mpt5 negatively regulates expression of the HO gene. Loss of MPT5 increases expression of reporter genes integrated into the ho locus, whereas overexpression of MPT5 decreases expression. Repression requires the 3'-UTR of HO, which contains a tetranucleotide, UUGU, also found in the binding sites of Pum and FBF. Mutation of UUGU to UACU in the HO 3'-UTR abolishes Mpt5-mediated repression. Studies using a three-hybrid assay for RNA binding indicate that Mpt5 binds to the 3'-UTR of HO mRNA containing a UUGU sequence but not a UACU sequence. These observations suggest that the yeast Puf homolog, Mpt5, negatively regulates HO expression post-transcriptionally (Tadauchi, 2001).

The Caenorhabditis elegans FBF protein and its Drosophila relative, Pumilio, define a large family of eukaryotic RNA-binding proteins. By binding regulatory elements in the 3' untranslated regions (UTRs) of their cognate RNAs, FBF and Pumilio have key post-transcriptional roles in early developmental decisions. In C. elegans, FBF is required for repression of fem-3 mRNA to achieve the hermaphrodite switch from spermatogenesis to oogenesis. FBF and NANOS-3 (NOS-3), one of three C. elegans Nanos homologs, interact with each other in both yeast two-hybrid and in vitro assays. The portions of each protein required for this interaction have been delineated. Worms lacking nanos function were derived either by RNA-mediated interference (nos-1 and nos-2) or by use of a deletion mutant (nos-3). The roles of the three nos genes overlap during germ-line development. In certain nos-deficient animals, the hermaphrodite sperm-oocyte switch is defective, leading to the production of excess sperm and no oocytes. In other nos-deficient animals, the entire germ line dies during larval development. This germ-line death does not require CED-3, a protease required for apoptosis. The data suggest that NOS-3 participates in the sperm-oocyte switch through its physical interaction with FBF, forming a regulatory complex that controls fem-3 mRNA. NOS-1 and NOS-2 also function in the switch, but do not interact directly with FBF. The three C. elegans nanos genes, like Drosophila nanos, are also critical for germ-line survival. It is proposed that this may have been the primitive function of nanos genes (Kraemer, 1999).

The nematode Caenorhabditis elegans has two sexes: males and hermaphrodites. Hermaphrodites Initially produce sperm but switch to producing oocytes. This switch appears to be controlled by the 3' untranslated region of fem-3 messenger RNA. A binding factor (FBF) has been identified that is a cytoplasmic protein that binds specifically to the regulatory region of fem-3 3'UTR and mediates the sperm/oocyte switch. The RNA-binding domain of FBF consists of a stretch of eight tandem repeats and two short flanking regions. This structural element is conserved in several proteins including Drosophila Pumilio, a regulatory protein that controls pattern formation in the fly by binding to a 3'UTR. It is proposed that FBF and Pumilio are members of a widespread family of sequence-specific RNA-binding proteins (Zhang, 1997).

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, it has been 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 have shown in vivo interaction between Xenopus Pumilio and CPE-binding protein (CPEB: Drosophila homolog Orb), 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 (Nakahata, 2001).

Drosophila Pumilio and a C. elegans Pumilio homolog, FBF, are members of the Pumilio-homology domain (Pum-HD) family, also known as thePuf (for Pumilio and FBF) family. The sequence in the C-terminal region of the Pum-HD family is highly conserved in many species, including human homologs (DDBJ/EMBL/GenBankTM accession numbers KIAA0099 and KIAA0235) deduced from their cDNAs. A Xenopus 2.0-kb sequence (DDBJ/EMBL/GenBankTM accession number AB045628) was obtained that contained a domain equivalent to those of Drosophila Pumilio (78% identity) and the human homolog KIAA0099 (95% identity). This domain is known as the diagnostic hallmark of the Pum-HD family and is defined by the presence of eight copies of an imperfect repeat sequence, comprising a specific RNA-binding domain (Nakahata, 2001).

The actual biological roles of XPum are completely unknown at present, but it can be speculated that XPum plays an important role in translational control of cyclin B1 mRNA, as in Drosophila. CPEB directly binds to maskin, a protein that can also bind directly to the cap-binding translation initiation factor elF-4E, which leads to translational repression. The dissociation of maskin from elF-4E allows elF-4G to bind to elF-4E, which brings elF-3 and the 40 S ribosomal subunit to the mRNA to initiate translation via cap-ribose methylation. Recent studies have also shown that a progesterone-induced early phosphorylation of CPEB at serine 174 is catalyzed by Eg2 and that this phosphorylation recruits cleavage and polyadenylation specificity factor into an active cytoplasmic polyadenylation complex. Thus, CPEB plays a key role in both translational repression and activation of mRNAs stored in oocytes. XPum is physically associated with CPEB in oocytes. In cooperation with CPEB, XPum may control the CPEB/maskin-mediated translational masking and unmasking to assure the highly coordinated successive translational activation of masked mRNAs during oocyte maturation. Further studies are required to understand the biological significance of the interactions among XPum, CPEB, and cyclin B1 mRNA, as well as to elucidate the functions of XPum in oocytes (Nakahata, 2001).

Germline stem cells are defined by their unique ability to generate more of themselves as well as differentiated gametes. The molecular mechanisms controlling the decision between self-renewal and differentiation are central unsolved problems in developmental biology with potentially broad medical implications. In Caenorhabditis elegans, germline stem cells are controlled by the somatic distal tip cell. FBF-1 and FBF-2, two nearly identical proteins, which together are called FBF ('fem-3 mRNA binding factor'), were originally discovered as regulators of germline sex determination. FBF also controls germline stem cells: in an fbf-1 fbf-2 double mutant, germline proliferation is initially normal, but stem cells are not maintained. It is suggested that FBF controls germline stem cells, at least in part, by repressing gld-1, which itself promotes commitment to the meiotic cell cycle. FBF belongs to the PUF family ('Pumilio and FBF') of RNA-binding proteins. Pumilio controls germline stem cells in Drosophila females, and, in lower eukaryotes, PUF proteins promote continued mitoses. It is suggested that regulation by PUF proteins may be an ancient and widespread mechanism for control of stem cells (Crittenden, 2002).

Protein synthesis of cyclin B by translational activation of the dormant mRNA stored in oocytes is required for normal progression of maturation. In Xenopus it has been shown that the cytoplasmic polyadenylation element (CPE) in the 3'-untranslated region (UTR) of cyclin B1 mRNA is responsible for both translational repression (masking) and activation (unmasking) of the mRNA (Mendez and Richter, 2001; Richter, 2000). The CPE is bound by a CPE-binding protein. In this study, the involvement of Xenopus Pumilio (XPum), a cyclin B1 mRNA-binding protein, was investigated in mRNA-specific translational activation. XPum exhibits high homology to mammalian counterparts, with amino acid identity close to 90%, even if the conserved RNA-binding domain is excluded. XPum is bound, in mature oocytes, to the unphosphorylated form of cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) through the RNA-binding domain. In addition to the CPE, the XPum-binding sequence of cyclin B1 mRNA acts as a cis-element for translational repression. Injection of anti-XPum antibody accelerated oocyte maturation and synthesis of cyclin B1, and, conversely, over-expression of XPum retarded oocyte maturation and translation of cyclin B1 mRNA, which was accompanied by inhibition of poly(A) tail elongation. The injection of antibody and the over-expression of XPum, however, had no effect on translation of Mos mRNA, which also contains the CPE. These findings provide the first evidence that XPum is a translational repressor specific to cyclin B1 in vertebrates. It is proposed that in cooperation with the CPEB-maskin complex, the master regulator common to the CPE-containing mRNAs, XPum acts as a specific regulator that determines the timing of translational activation of cyclin B1 mRNA by its release from phosphorylated CPEB during oocyte maturation (Nakahata, 2003).

One possible mechanism of translational activation of cyclin B1 mRNA is that a dissociation of XPum from phosphorylated CPEB during oocyte maturation induces destabilization of the CPEB-maskin-eIF4E complex and provides a cue that leads to unmasking of cyclin B1 mRNA by the mechanism common to CPE-containing mRNAs. In this respect, it is noteworthy that phosphorylation of CPEB on Ser210, which occurs about the time of cyclin B1 translation, is sufficient for selective translational activation of cyclin B1. While this phenomenon has been explained in relation to degradation of CPEB, it is also conceivable that the later phosphorylation of CPEB induces release of XPum from the CPEB-maskin-eIF4E complex and that this event triggers translational activation of cyclin B1. Consistent with this possibility, it has been demonstrated that phosphorylation of CPEB is required for its dissociation from a large ribonucleoprotein complex upon oocyte maturation, prior to degradation (Nakahata, 2003).

date revised: 2 December 99

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

Please e-mail comments/corrections to brodyt@codon.nih.gov