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

Yeast Pumilio 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).

In yeast Saccharomyces cerevisiae, Ash1p, a protein determinant for mating-type switching, is segregated within the daughter cell nucleus to establish asymmetry of HO expression. The accumulation of Ash1p results from ASH1 mRNA that is sorted as a ribonucleoprotein particle (mRNP or locasome) to the distal tip of the bud where translation occurs. To study the mechanism regulating ASH1 mRNA translation, the ASH1 locasome was isolated and the associated proteins were characterized by MALDI-TOF. One of these proteins was Puf6p, a new member of the PUF family of highly conserved RNA-binding proteins such as Pumilio in Drosophila, responsible for translational repression, usually to effect asymmetric expression. Puf6p binds PUF consensus sequences in the 3'UTR of ASH1 mRNA and represses the translation of ASH1 mRNA both in vivo and in vitro. In the puf6Delta strain, asymmetric localization of both Ash1p and ASH1 mRNA were significantly reduced. It is proposed that Puf6p is a protein that functions in the translational control of ASH1 mRNA, and this translational inhibition is necessary before localization can proceed (Gu, 2004).

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).

PUF proteins, a family of RNA-binding proteins, interact with the 3' untranslated regions (UTRs) of specific mRNAs to control their translation and stability. PUF protein action is commonly correlated with removal of the poly(A) tail of target mRNAs. This study focuses on how PUF proteins enhance deadenylation and mRNA decay. A yeast PUF protein physically binds Pop2p (Drosophila homolog Pop2), which is a component of the Ccr4p-Pop2p-Not deadenylase complex, and Pop2p is required for PUF repression activity. By binding Pop2p, the PUF protein simultaneously recruits the Ccr4p deadenylase (homolog of Drosophila Twin) and two other enzymes involved in mRNA regulation, Dcp1p and Dhh1p. Regulated deadenylation was reconstituted in vitro and it was demonstrated that the PUF-Pop2p interaction is conserved in yeast, worms and humans. It is suggested that the PUF-Pop2p interaction underlies regulated deadenylation, mRNA decay and repression by PUF proteins (Goldstrohm, 2006).

Invertebrate Pumilio homologs

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).

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).

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).

In the C. elegans germline, GLP-1/Notch signaling and two nearly identical PUF (Pumilio and FBF) protein family RNA binding proteins, FBF-1 and FBF-2, promote proliferation. Here, the fbf-1 and fbf-2 genes are largely redundant for promoting mitosis but they have opposite roles in fine-tuning the size of the mitotic region. The mitotic region is smaller than normal in fbf-1 mutants but larger than normal in fbf-2 mutants. Consistent with gene-specific roles, fbf-2 expression is limited to the distal germline, while fbf-1 expression is broader. The fbf-2 gene, but apparently not fbf-1, is controlled by GLP-1/Notch signaling, and the abundance of FBF-1 and FBF-2 proteins is limited by reciprocal 3′UTR repression. It is proposed that the divergent fbf genes and their regulatory subnetwork enable a precise control over size of the mitotic region. Therefore, fbf-1 and fbf-2 provide a paradigm for how recently duplicated genes can diverge to fine-tune patterning during animal development (Lamont, 2004).

RNA binding proteins are key regulators of the germline decision between proliferation and differentiation. Of particular importance to this paper are FBF-1 and FBF-2 (for fem-3 Binding Factor) -- two nearly identical regulators of the PUF family. The FBF-1 and FBF-2 proteins are collectively called FBF, and similarly, fbf-1 and fbf-2 are collectively called the fbf genes. The nucleotide sequences of fbf-1 and fbf-2 are 93% identical, and the amino acid sequences are 91% identical, suggesting that fbf-1 and fbf-2 are recently duplicated genes. During early larval stages, germline proliferation is normal in fbf-1 fbf-2 double mutants, but in the fourth larval stage, the germline precociously leaves the mitotic cell cycle to enter meiosis and differentiate as sperm. In addition, depletion of both fbf-1 and fbf-2 eliminates the hermaphrodite switch from spermatogenesis to oogenesis. Therefore, FBF is required for continued mitotic divisions in the germline as well as for the hermaphrodite sperm/oocyte switch (Lamont, 2004).

PUF proteins bind specifically to regulatory elements, usually in the 3' untranslated region (UTR) of a target mRNA, and repress that mRNA, either by promoting mRNA degradation or inhibiting translation. Pumilio, for example, inhibits translation of hunchback mRNA in the early Drosophila embryo, whereas PUF-5/Mpt5 destabilizes HO mRNA in yeast. In C. elegans, FBF-1 and FBF-2 promote mitosis by repressing mRNAs that encode regulators critical for entry into the meiotic cell cycle, and they promote the sperm/oocyte switch by repressing the fem-3 sperm-promoting mRNA. Both FBF-1 and FBF-2 bind specifically to the same RNA target sequence, which differs from the Pumilio binding site. The molecular mechanism by which FBF represses mRNAs in the C. elegans germline remains unknown, but by analogy with its homologs in yeast and Drosophila, FBF is likely to control the stability or translation of its target mRNAs (Lamont, 2004).

Previous studies have suggested that FBF-1 and FBF-2 are redundant: fbf-1 single mutants are grossly normal, albeit with smaller mitotic regions and more hermaphrodite sperm than wild-type. This study confirms the fbf-1/fbf-2 redundancy but also identify individual roles for each gene in regulating the size of the mitotic region. Like fbf-1, the fbf-2 single mutants are grossly normal, but in contrast to fbf-1, fbf-2 mutant germlines have a larger mitotic region than normal and can be feminized. Consistent with fbf-1 and fbf-2 having individual roles, their mRNAs and proteins are expressed in distinct patterns. Furthermore, the fbf-2 gene appears to be a direct target of GLP-1/Notch signaling, a finding that forges the first molecular link between GLP-1/Notch signaling and the RNA regulatory circuit. fbf-1 and fbf-2 repress each other's expression and this reciprocal repression is likely to be direct via FBF binding sites in the fbf-1 and fbf-2 3' UTRs. It is suggested that GLP-1/Notch signaling and FBF autoregulation work together to control the distribution and amount of FBF and thereby fine-tune the size of the mitotic region (Lamont, 2004).

Since stem cells are rare and difficult to study in vivo in adults, the use of classical models of regeneration to address fundamental aspects of the stem cell biology is emerging. Planarian regeneration, which is based upon totipotent stem cells present in the adult – the so-called neoblasts – provides a unique opportunity to study in vivo the molecular program that defines a stem cell. The choice of a stem cell to self-renew or differentiate involves regulatory molecules that also operate as translational repressors, such as members of PUF proteins. In this study, a homologue of the Drosophila PUF gene Pumilio (DjPum) was identified in the planarian Dugesia japonica, with an expression pattern preferentially restricted to neoblasts. Through RNA interference (RNAi), gene silencing of DjPum was demonstrated to dramatically reduces the number of neoblasts, thus supporting the intriguing hypothesis that stem cell maintenance may be an ancestral function of PUF proteins (Salvetti, 2005).

Xenopus Pumilio homologs

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).

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).

Mammalian Pumilio homologs

Puf proteins are developmental regulators that control mRNA stability and translation by binding sequences in the 3' untranslated regions of their target mRNAs. The structure of the RNA binding domain of the human Puf protein, Pumilio1, has been determined, bound to a high-affinity RNA ligand. The RNA binds the concave surface of the molecule, where each of the protein's eight repeats makes contacts with a different RNA base via three amino acid side chains at conserved positions. These three side chains were mutated in one repeat, thereby altering the sequence specificity of Pumilio1. Thus, the high affinity and specificity of the PUM-HD for RNA is achieved using multiple copies of a simple repeated motif (Wang, 2002).

Germ cell development is complex; it encompasses specification of germ cell fate, mitotic replication of early germ cell populations, and meiotic and postmeiotic development. Meiosis alone may require several hundred genes, including homologs of the BOULE (BOL) and PUMILIO (PUM) gene families. Both BOL and PUM homologs encode germ cell specific RNA binding proteins in diverse organisms where they are required for germ cell development. Human BOL forms homodimers and is able to interact with a PUMILIO homolog, PUM2. The domain of BOL that is required for dimerization and for interaction with PUM2 was mapped. BOL and PUM2 can form a complex on a subset of PUM2 RNA targets that is distinct from targets bound by PUM2 and another deleted in azoospermia (DAZ) family member, DAZ-like (DAZL). This suggests that RNA sequences bound by PUM2 may be determined by protein interactions. This data also suggests that although the BOL, DAZ, and DAZL proteins are all members of the same gene family, they may function in distinct molecular complexes during human germ cell development (Urano, 2005).

Members of the Pumilio and DAZL family of RNA binding proteins are required for germ cell development in Drosophila, Xenopus, and Caenorhabditis elegans. This study reports identification and characterization of RNA sequences to which PUM2 and DAZL bind. Human PUM2 specifically recognizes the Drosophila Pumilio RNA target (the NRE or Nanos regulator element sequence); single nucleotide changes in the NRE abolished PUM2 binding. Then, coimmunoprecipitation was used to isolate human transcripts specifically bound by PUM2 and DAZL and subsequently those were identified that contain NRE-like sequence elements. The interacting proteins, PUM2 and DAZL, are capable of binding the same RNA target and mRNA sequences bound by both proteins in the 3'UTR of human SDAD1 mRNA were further characterized. Taken together, the results define sequences to which these germ cell-specific RNA binding proteins may bind to promote germ cell development (Fox, 2005).

Pumilio (Pum) protein acts as a translational inhibitor in several organisms including yeast, Drosophila, Xenopus, and mammals. Two Pumilio genes, Pum1 and Pum2, have been identified in mammals, but their function in neurons has not been identified. In this study, it was found that Pum2 mRNA is expressed during neuronal development and that the protein is found in discrete particles in both the cell body and the dendritic compartment of fully polarized neurons. This finding indicates that Pum2 is a novel candidate of dendritically localized ribonucleoparticles (RNPs). During metabolic stress, Pum2 is present in stress granules (SGs), which are subsequently detected in the somatodendritic domain. It remains excluded from processing bodies under all conditions. When overexpressed in neurons and fibroblasts, Pum2 induces the formation of SGs that also contain T-cell intracellular antigen 1 (TIA-1)-related protein, eukaryotic initiation factor 4E, poly(A)-binding protein, TIA-1, and other RNA-binding proteins including Staufen1 and Barentsz. This induction of SGs is dependent on the RNA-binding domain and a glutamine-rich region in the N terminus of Pum2. This glutamine-rich region behaves in a similar manner as TIA-1 and prion protein, two molecules with known roles in protein aggregation. Pum2 downregulation in neurons via RNA interference (RNAi) interferes with the formation of SGs during metabolic stress. Cotransfection with an RNAi-resistant portion of the Pum2 mRNA restores SG formation. These results suggest a role for Pum2 in dendritic RNPs and SG formation in mammalian neurons (Vessey, 2006).

date revised: 2 December 99

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