In the Drosophila ovary, germline stem cell (GSC) self-renewal is controlled by both extrinsic and intrinsic factors. The Bmp signal from niche cells controls GSC self-renewal by directly repressing a Bam-dependent differentiation pathway in GSCs. pelota (pelo), which has been previously shown to be required for Drosophila male meiosis, was identified in a genetic screen as a dominant suppressor of the dpp overexpression-induced GSC tumor phenotype. Pelo acts in controlling GSC self-renewal by repressing a Bam-independent differentiation pathway. In pelo mutant ovaries, GSCs are lost rapidly owing to differentiation. Results from genetic mosaic analysis and germ cell-specific rescue show that it functions as an intrinsic factor to control GSC self-renewal. In pelo mutant GSCs, Bmp signaling activity detected by Dad-lacZ expression is downregulated, but bam expression is still repressed. Furthermore, bam mutant germ cells are still able to differentiate into cystocytes without pelo function, indicating that Pelo is involved in repressing a Bam-independent differentiation pathway. Consistent with its homology to the eukaryotic translation release factor 1alpha, Pelo is shown to be localized to the cytoplasm of the GSC. Therefore, Pelo controls GSC self-renewal by repressing a Bam-independent differentiation pathway possibly through regulating translation. Since Pelo is highly conserved from Drosophila to mammals, it may also be involved in the regulation of adult stem cell self-renewal in mammals, including humans (Xi, 2005).

Stem cells are characterized by their ability to self-renew and generate differentiated cells throughout the lifetime of an organism. Understanding how stem cell self-renewal is controlled is an important issue in stem cell biology and will help realize the potentials inherent in stem cell-based therapies. Studies from diverse systems indicate that stem cell self-renewal is controlled by both extrinsic factors (niche signals) and intrinsic factors. Niche signals have been shown to control GSC self-renewal by directly repressing expression of differentiation-promoting genes in the Drosophila ovary (Chen, 2003a; Song, 2004). Therefore, the identification of pathways and genes that repress stem cell differentiation is crucial for understanding how stem cell self-renewal is controlled (Xi, 2005).

In the Drosophila ovary, GSCs reside in a structure called the germarium, which is at the anterior end of an ovariole (Lin, 2002; Xie, 2001). At the anterior tip of the germarium, three types of somatic cells (terminal filament cells, cap cells and inner sheath cells) constitute a niche that supports two or three GSCs (Lin, 2002; Xie, 2001; Xie, 2000). One GSC divides to generate two daughter cells: the daughter cell maintaining contact with the cap cells through DE-cadherin-mediated cell adhesion renews itself as a stem cell, while the daughter cell moving away from the cap cells differentiates into a cystoblast (Song, 2002b). The cystoblast divides four times with incomplete cytokinesis to form a 16-cell cyst in which one cell becomes an oocyte and the rest become nurse cells. The Bmp signals that are produced by the niche, Dpp and Gbb, have essential roles in controlling GSC self-renewal: reduction of Bmp signaling activity results in the loss of GSCs by differentiation; overexpression of dpp in the germarium produces GSC-like tumors (Song, 2004; Xie, 1998). Bmps from the cap cells function as short-range signals that directly repress the transcription of bam in GSCs to maintain their self-renewal, and also allow cystoblasts lying one cell diameter away to differentiate (Chen, 2003a; Song, 2004). bam is necessary and sufficient for germ cell differentiating in the Drosophila ovary. In addition, two other genes, Yb and piwi, function in the somatic niche cells to control GSC (Cox, 2000; King, 2001). Yb encodes a novel protein and directly regulates expression of piwi and hh in TFs; hh signaling also modulates GSC self-renewal though it is not essential (King, 2001). piwi encodes a family of conserved RNA-binding proteins and is required in the niche cells for controlling GSC self-renewal and inside GSCs for their division. Two recent studies have shown that piwi also maintains GSC self-renewal by repressing bam expression through regulation of either the Bmp signaling pathway or a Bmp-independent signaling pathway (Chen, 2005; Szakmary, 2005). However, it remains unclear how piwi controls GSC division intrinsically (Xi, 2005).

Two translational repressors, Nanos (Nos) and Pumilio (Pum), have been shown to be required for the maintenance of ovarian GSCs by preventing differentiation. Pum/Nos repress differentiation of PGCs and GSCs through a Bmp-independent pathway; their expression is not regulated by Bmp signaling and their mutations cannot suppress hyperactive Bmp signaling-induced PGC proliferation. It is likely that Nos and Pum are involved in repressing translation gene products that are important for germ cell differentiation and thereby for controlling GSC self-renewal. To identify further intrinsic factors that are required for Bmp-mediated GSC self-renewal, a genetic screen was performed to identify dominant suppressors of the dpp-induced GSC-like tumor phenotype. One of the suppressors is pelota (pelo), which has been studied for its role in the regulation of Drosophila male meiosis. Cellular and molecular analysis showed that pelo is required for the progression through meiosis in spermatogenesis and encodes an evolutionarily conserved protein that contains a eukaryotic release factor 1 alpha (eRF1alpha)-like domain at its C terminus (Eberhart, 1995). The studies on the yeast pelo homolog dom34 suggest that Pelo is involved in translational regulation. Deletion of Dom34 causes growth retardation, defective sporulation and reduces polyribosomes (Davis, 1998). dom34 has a strong genetic interaction with RPS30A, which encodes ribosomal protein S30A; overexpression of RPS30A rescues the growth defects and reduced polyribosomes of dom34 mutants (Davis, 1998). Moreover, Dom34 specifically interacts with Hbs1, a small GTPase that is also implicated in translational regulation (Carr-Schmid, 2002). It has been recently shown that pelo knockout mice exhibit early embryonic lethality with defects in cell division and proliferation (Adham, 2003). Taken together, pelo may be involved in the regulation of meiosis and mitosis possibly through regulating translation. In this study, an unexpected new role has been discovered for Pelo in the control of GSC self-renewal and division in the Drosophila ovary, possibly through regulating translation (Xi, 2005).

It has been shown that bam is both essential and sufficient for cystoblast differentiation. The observation that pelo¹ mutant GSCs are lost because of differentiation but do not upregulate bam expression suggests that pelo mutant GSCs differentiate using a bam-independent pathway. If so, it should be expected that bam mutant germ cells would be able to differentiate in the absence of pelo function. To test this idea, the genetic relationship between bam and pelo was investigated. The pelo¹ homozygous GSCs that were heterozygous for bamDelta86, a deletion allele of bam, were still lost rapidly as in the pelo¹ mutant GSCs; the bamDelta86 homozygous germ cells that were also heterozygous for pelo¹ still failed to differentiate, as did the bamDelta86 mutant one. Interestingly, in pelo¹; bamDelta86 double homozygous germaria, most of the germ cells were cysts with branched fusomes, and some of them still retained a round spectrosome. Unusually, the round spectrosomes in the remaining single germ cells were larger than those in bam mutant single germ cells, suggesting the single germ cells could be growth-arrested cystoblasts but can continue to grow their spectrosome. The morphology of the branched fusomes of the double mutant cysts appeared abnormal. To further determine whether the oocytes form in these pelo; bam mutant cysts, the expression of Orb protein was examined in the double mutant germaria. Orb normally starts to accumulate in newly formed wild-type oocytes; however, no obvious Orb expression was detected in the pelo; bam mutant germaria, indicating that there is no oocyte formation in the double mutant cysts. Since pelo mutant cysts can still form the oocyte, bam is probably required late for oocyte formation. These findings show that pelo mutant cystoblasts can differentiate without functional bam, and further suggest that pelo must repress a bam-independent differentiation pathway to maintain GSC self-renewal (Xi, 2005).

This study has revealed a new function of pelo in controlling GSC self-renewal in the Drosophila ovary. Genetic clonal analysis and rescue experiment results show that Pelo is required intrinsically for controlling GSC self-renewal. It is involved in the regulation of some Bmp response in GSCs but is not required for repressing bam expression. Genetic analysis further indicates that it controls GSC self-renewal by repressing a bam-independent differentiation pathway. Although translational control has been implicated in the regulation of GSC self-renewal, this is the first time it has been shown that a translational release factor-like protein controls GSC self-renewal. Since Pelo is highly conserved from Drosophila to human, it is also possible that its mammalian homologs might also be involved in the control of stem cell self-renewal (Xi, 2005).

Drosophila Pelo belongs to a family of evolutionarily conserved proteins with their primary function in the regulation of cell division. In the yeast, dom34 (pelo) mutant cells grow slowly and have defects in the entry of meiosis, indicating that it is required for mitosis and meiosis. In mice, disruption of the pelo gene causes early embryonic lethality and defects in cell cycle progression (Adham, 2003). Although pelo is ubiquitously expressed throughout Drosophila development (Eberhart, 1995), the pelo mutants survive to adulthood without obvious defects in the body. In Drosophila, pelo has been shown to be required to control meiotic cell cycle progression in male germ cells. In this study, pelo has been shown to be required intrinsically for controlling self-renewal and division of GSCs but not SSCs in the ovary; this requirement is supported by rescue and stem cell clonal analysis experiments. Even though Pelo members are required for regulating cell cycle progression from yeast to mammals, it remains unclear how they accomplish this function (Xi, 2005).

The only clue to the potential cellular function of Pelo comes from its high homology to the translation release factor 1. Its likely function as a translational regulator is further complicated by the presence of a highly conserved NLS sequence (Eberhart, 1995). Using an epitope-tagged Pelo that can rescue pelo mutants, it was demonstrated that Pelo is mainly localized to the cytoplasm of both S2 cells and germ cells. Furthermore, the pelo gene with a mutated putative NLS is still fully functional. The yeast Pelo, Dom34, is also localized to the cytoplasm (Davis, 1998; Huh, 2003). These findings support the idea that Pelo proteins function in the cytoplasm as translational regulators in different organisms. If Pelo protein truly functions as a translational release factors,it must directly interact with ribosomes that are either associated with ER or in the cytoplasm. Consistent with this idea, some Pelo proteins are associated with ER membranes though the majority of Pelo proteins are not associated with ER membranes. In yeast, dom34 mutants have dramatically reduced polyribosomes and can be rescued by a high-copy of the ribosomal protein S30A gene, indicating that Dom34 is involved in translation (Davis, 1998). In addition, expression of Drosophila pelo in dom34 mutants can rescue growth defects, indicating its conserved function during evolution. Therefore, Pelo is also likely a translational regulator in Drosophila, and is involved in regulating translation of a specific class of mRNAs that are important for germ cell function. In the future, it will be important to investigate whether Pelo is indeed involved in translational regulation and to identify its targets in germ cells (Xi, 2005).

Before this work, pelo had not been shown to be involved in regulating any signaling pathways in any organisms. The Bmp pathway is a major signaling pathway that is essential for controlling GSC self-renewal and division in the Drosophila ovary (Song, 2004; Xie, 1998). The Bmp signaling activities can be reliably monitored by expression of Dad in GSCs. It is anticipated that pelo must somehow interact with the Bmp pathway in controlling GSC self-renewal, since pelo was also identified as a dominant suppressor of Dpp overexpression-induced GSC-like tumors. In this study, GSCs mutant for pelo are shown to downregulate Dad. These findings indicate that Pelo participates in Bmp signaling to control expression of dpp target genes in GSCs such as Dad (Xi, 2005).

In the Drosophila ovary, one of the ways in which Bmp signaling controls GSC self-renewal is to directly repress bam expression in GSCs. bam is necessary and sufficient for cystoblast differentiation in the Drosophila ovary. In this study, Pelo is shown to be essential for controlling GSC self-renewal but is not involved in repressing bam expression. pelo mutant GSCs have normal bam repression but their progeny can still differentiate without bam, suggesting that pelo maintains GSCs by repressing a bam-independent pathway. During the preparation of this manuscript, two studies were published showing that pum controls GSC self-renewal by repressing a bam-independent pathway (Chen, 2005; Szakmary, 2005). Pum is known to work together with Nos, which is also essential for Drosophila ovarian GSC self-renewal, to repress gene translation in the embryo. Since Pum/Nos does not participate in Bmp signaling and Pelo is a translational release factor-like protein, it is proposed that Pelo works in a parallel genetic pathway with Pum in repressing the same or different Bam-independent differentiation pathways through regulating translation. Although it is essential for repressing a Bam-independent pathway(s) in GSCs, Pelo is not so sufficient for doing so; Bmp signaling functions to repress bam, as evidenced by the observation that overexpression of pelo has no effect on the GSC maintenance and differentiation. In the future, it will be important to molecularly and genetically characterize the Bam-independent pathway repressed by Pelo and to further understand how Pelo represses it in relation to Pum (Xi, 2005).

GENE STRUCTURE

cDNA clone length - 2028 bp

Bases in 5' UTR - 255

Exons - 5

Bases in 3' UTR - 585

PROTEIN STRUCTURE

Amino Acids - 395

Structural Domains

The Pelota protein sequence was compared to protein sequences in the NCBI database using the BLASTP program. This search resulted in two significant matches: S. cerevisiae DOM34 and C. elegans R74.6. Dom34 has been described by an analysis of sequences duplicated between yeast chromosomes 3 and 14 and is 33% identical to Pelota with only one gap. R74.6 was identified in the C. elegans genome sequencing effort; it is 58% identical to Pelota, with no gaps and an identically positioned start methionine. All three proteins feature a potential nuclear localization sequence in the N terminal half, as well as an acidic domain at the carboxyl terminus. Searches of nucleotide databases translated in six frames using the PBLASTN program revealed three cDNA expressed sequence tags (ESTs) encoding predicted proteins with high identity to Pelota. Two H. sapiens polypeptide sequences, 110 and 93 amino acids in length, and a 96 amino acid A. thaliana polypeptide are 70%, 53% and 55% identical to Pelota, respectively. The two human sequences are nonoverlapping, but may represent portions of a single Pelota homolog, since one contains the conserved nuclear localization signal and the other the carboxyl terminal acidic domain (Eberhart, 1995).

EVOLUTIONARY HOMOLOGS

Pelota homologs in bacteria and yeast

An open reading frame (pelA) specifying a homolog of pelota and DOM34, proteins required for meiotic cell division in Drosophila melanogaster and Saccharomyces cerevisiae, respectively, has been cloned, sequenced and identified from the archaebacterium Sulfolobus solfataricus. The S. solfataricus PelA protein is about 20% identical with pelota, DOM34 and the hypothetical protein R74.6 of Caenorhabditis elegans. The presence of a pelota homolog in archaebacteria implies that the meiotic functions of the eukaryotic protein were co-opted from, or added to, other functions existing before the emergence of eukaryotes. The nuclear localization signal and negatively charged carboxy-terminus characteristic of eukaryotic pelota-like proteins are absent from the S. solfataricus homolog, and hence may be indicative of the acquired eukaryotic function(s) (Ragan, 1996).

The DOM34 gene of Saccharomyces cerevisiae is similar to genes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminished. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34delta growth defect. dom34delta mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34delta mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family (Davis, 1998).

G proteins, which bind and hydrolyze GTP, are involved in regulating a variety of critical cellular processes, including the process of protein synthesis. Many members of the subfamily of elongation factor class G proteins interact with the ribosome and function to regulate discrete steps during the process of protein synthesis. Despite sequence similarity to factors involved in translation, a role for the yeast Hbs1 protein has not been defined. In this work, a genetic relationship has been identified between genes encoding components of the translational apparatus and HBS1. HBS1, while not essential for viability, is important for efficient growth and protein synthesis under conditions of limiting translation initiation. The identification of an Hbs1p-interacting factor, Dom34p, which shares a similar genetic relationship with components of the translational apparatus, suggests that Hbs1p and Dom34p may function as part of a complex that facilitates gene expression. Dom34p contains an RNA binding motif present in several ribosomal proteins and factors that regulate translation of specific mRNAs. Thus, Hbs1p and Dom34p may function together to help directly or indirectly facilitate the expression either of specific mRNAs or under certain cellular conditions (Carr-Schmid, 2002).

Pelota homolog in mammals

The pelota gene of Drosophila encodes a protein that was found to be included in cell cycle regulation. Mutations were found to result in spermatogenic arrest, female sterility and disturbances in the patterning of the eye. The human pelota cDNA (PELO) encodes a 385-amino-acid protein. Southern blot and fluorescence in situ hybridization analyses revealed that PELO is present as a single copy gene in the human genome and is localized on chromosome 5q11.2. Northern blot analysis revealed the presence of a 1.6-kb transcript in all tissues studied and an additional 2.0-kb transcript in testis (Shamsadin, 2000).

The pelota gene of Drosophila encodes a protein which is included in cell cycle regulation. Mutations were found to result in spermatogenic arrest, female sterility and disturbances in the patterning of the eye. cDNA clones coding for the human pelota gene (PELO) have been identified. The murine pelota cDNA and gene (Pelo) encodes a 385-amino-acid protein. The exon-intron structure of the gene, which contains three exons, was determined. Comparison of the mouse amino acid sequences with the human and Drosophila sequences revealed an overall high identity (96% and 70%, respectively). Northern blot analysis detected a 1.7-kb transcript in all tissues studied. Southern blot analyses revealed that the pelota gene is present as a single copy in the mouse genome. The mouse pelota gene (Pelo) was mapped to the distal end of chromosome 13, in a region that is homologous with a segment of human chromosome 5q11 containing the orthologous human gene. Cloning of the mouse gene is an important step to study the function of the pelota gene in mammals and to create a mouse model for this evolutionarily conserved gene (Shamsadin, 2002).

Mutations in either the Drosophila pelota or pelo gene or the Saccharomyces cerevisiae homologous gene, DOM34, cause defects of spermatogenesis and oogenesis in Drosophila, and delay of growth and failure of sporulation in yeast. These phenotypes suggest that pelota is required for normal progression of the mitotic and meiotic cell cycle. To determine the role of the pelota in mouse development and progression of cell cycle, a targeted disruption of the mouse PELO was established: heterozygous animals are variable and fertile. Genotyping of the progeny of heterozygous intercrosses shows the absence of Pelo^-/- pups and suggests an embryo-lethal phenotype. Histological analyses reveal that the homozygous Pelo deficient embryos fail to develop past day 7.5 of embryogenesis (E7.5). The failure of mitotic active inner cell mass of the Pelo^-/- blastocysts to expand in growth after 4 days in culture and the survival of mitotic inactive trophoplast indicate that the lethality of Pelo-null embryos is due to defects in cell proliferation. Analysis of the cellular DNA content reveals the significant increase of aneuploid cells in Pelo^-/- embryos at E7.5. Therefore, the percent increase of aneuploid cells at E7.5 may be directly responsible for the arrested development and suggests that Pelo is required for the maintenance of genomic stability (Adham, 2003).

date revised: 30 March 2006

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