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Gene name - piwi Synonyms - Cytological map position - 32C--32C Function - regulates asymmetric cell division Keywords - oogenesis, stem cell renewal, posttranscriptional RNA-based gene silencing, Repeat-associated small interfering RNAs (rasiRNAs), retrotransposon silencing |
Symbol - piwi FlyBase ID:FBgn0004872 Genetic map position - 2-[40] Classification - Argonaute family protein Cellular location - nuclear |
The Drosophila piwi gene is required for the asymmetric division of germ-line stem cells (GSCs) that takes place during the process of gametogenesis. GSCs are the source of gametes during gametogenesis. The process may be likened to an assembly line one can follow, from the raw material of the GSCs to the egg and sperm 'finished product'. Before a consideration of piwi, a brief review of gametogenesis is in order.
In Drosophila, germ line stem cells are present at the apical tip of the ovariole, the functional unit of the ovary. In the ovariole, GSCs are located in a specialized structure called the germarium, the structure in which the egg develops from germ line stem cells. In each germarium, two to three GSCs contact the somatic basal terminal filament cells. GSCs undergo oriented asymmetric divisions producing two daughter cells -- one is a daughter stem cell, which remains associated with the terminal filament; the other is a differentiated daughter, the cystoblast, which is displaced one cell away from the terminal filament. The germ-line cyst, derived from the cystoblast, is then enveloped by follicle cells that are produced by somatic stem cells to form an egg chamber, which buds off the germarium, joins pre-existing egg chambers in a linear array to constitute the ovariole, and will eventual develop into a mature egg. This assembly line organization, with each egg chamber representing a differentiated stem cell product whose position along the ovariole corresponds to its birth order, provides a unique opportunity for the study of stem cell division (Cox, 1998 and references).
Although piwi is expressed in the germ line, this expression is not involved in regulation of stem cell division. Instead, piwi expression in somatic cells is involved in this essential function. piwi expression in the germ line provides a maternal component for embryogenesis. Although Drosophila piwi is required for the asymmetric division of GSCs into daughter GSCs and daughter cystoblasts, piwi is not essential for the further differentiation of the cystoblast. The self-renewing ability of GSCs is controlled both by extrinsic signaling and by cell-autonomous mechanisms. piwi is not required cell autonomously in the germline, but piwi expression in adjacent somatic cells regulates GSC division (Cox, 1998).
piwi mutant ovaries contain a normal number of GSCs at the onset of oogenesis (late third instar larval stage); however, the mutation produces an equal or somewhat smaller number of gametes in the adult gonads, and they no longer contain GSCs (Lin, 1997). This failure of germ-line maintenance could be due to the following: (1) the differentiation of GSCs without self-renewing divisions; (2) a defect in the asymmetry of GSC division, producing aberrant germ cells that eventually degenerate; and/or (3) a secondary defect influenced by abnormal ovary differentiation. To examine whether the failure of germ-line maintenance is a secondary defect due to abnormal ovary development, the ovarian morphology of piwi mutants was examined. Mutant ovaries show normal morphology at the third instar larval stage. Their germ-line cells are normal in number and are correctly positioned along the medial plane of the ovary. Moreover, the expected number of terminal filaments are forming, so that at the pupal stage, the ovary differentiates normally, partitioning GSCs and their daughter cells correctly into individual germaria and ovarioles. GSCs are able to divide several times to provide a normal complement of germ cells to the germarium. Yet, mutant GSCs subsequently fail to continue self-renewing divisions, and the existing germ-line cysts often degenerate during the late pupal stage so that the adult ovarioles contain germaria lacking germ lines and fewer egg chambers than expected. These observations suggest that the failure of GSC maintenance in piwi mutants is not a secondary defect due to abnormal ovary development (Cox, 1998).
The main oogenic defect in piwi mutants is the differentiation of GSCs without self-renewing divisions immediately following the initiation of oogenesis. At this stage in wild-type ovaries, GSCs in 16-23 newly formed germaria have initiated asymmetric divisions to generate multiple developing germ-line cysts. However, in mutant ovaries, GSCs and their immediate differentiated daughters, cystoblasts, are undetectable, as indicated by the absence of spectrosome-containing germ cells (see Spectrin for the definitions of spectrosome and fusome). Instead, most ovaries contain differentiated germ-line cysts whose number approximately equals that of GSCs. These cysts are much larger in size and contain two- to multicell stage fusomes, indicating their differentiating state. By the adult stage, most ovarioles contain only two egg chambers, either normal or abnormal, derived from these cysts, but no other germ-line cells. This defect contrasts with the development of wild-type ovaries, in which ovarioles contain a fully developed germarium and a stage 1 egg chamber by the 48-hr pupal stage and have produced multiple egg chambers by the adult stage. This observation indicates that the mutant GSCs have differentiated into germ-line cysts without self-renewing divisions (Cox, 1998).
piwi is expressed in the somatic germarial terminal filament cells apical to the GSCs, in somatic follicle cells that surround the ovary, and in the germ-line cells that constitute the ovary. Theoretically, piwi could be required in either the germ-line cells or somatic cells for asymmetic cell division of the germ-line stem cells. To examine the roles of somatic and germ-line piwi in GSC asymmetric division, genetic clonal analyses were conducted on piwi1 and piwi2 mutations. Removing the piwi+ chromosome from the germ line allows oogenesis, including GSC division, to occur normally. Thus, the requirement of piwi for GSC division does not reside in the germ line but in somatic cells. To determine whether piwi is required in somatic cells outside the ovary for GSC maintenance, wild-type germaria were transplanted into the abdominal cavity of homozygous piwi mutant females. Transplanted germaria produce a normal number egg chambers after 7 days of incubation in mutant hosts. Thus wild-type germaria continue oogenesis at a normal rate in the piwi mutant females. Hence, piwi is not required in extra-ovarian cells for GSC maintenance. By inducing mitotic recombination at the second and third instar larval stages, piwi minus somatic clones were generated throughout ovarioles, with some egg chambers completely covered by piwi mutant follicle cells. These egg chambers developed normally, indicating that piwi function is not required in follicle cells for egg chamber development. Because follicle cells are derived from their precursor cells in region II of the germarium, this suggests that piwi is not required in somatic cells from germarial region II for GSC division and ovarian development. These results, together with the fact that piwi is expressed in the terminal filament cells at the tip of the germarium, suggest that piwi is required in these somatic cells in the anterior-most tip of the germarium to regulate GSC division (Cox, 1998).
Findings of piwi homologs in plants, C. elegans and vertebrates, and studies of their functions in various organisms suggest that piwi is involved in an evolutionarily conserved stem cell self-renewing mechanism. Among this class of genes, the significantly higher homology between piwi and human hiwi as compared with that between piwi and C. elegans prg-1/prg-2 (prg stands for piwi-related gene) suggests that hiwi function is closer to piwi. Consistent with this, GSC division and gametogenesis in humans are much more similar to the same processes in Drosophila than those in C. elegans, whose gonads contain syncytial mitotic germ-line nuclei that divide symmetrically and are capable of self-renewing only as a population. RNA interference (RNAi) experiments, which are known to mimic the effects of gene mutation, were carried out in C. elegans. RNAi by prg-1 double stranded RNA causes germ-line depletion similar to that in piwi mutants. This suggests that the piwi-mediated mechanism in germ-line self-renewal is conserved even in this evolutionarily distant organism without stereotypic GSCs. The conservation of the piwi-mediated mechanism appears to extend to the plant kingdom as well. The overall homology between Piwi and ZWILLE (ZLL) and AGO of Arabidopsis thaliana is worth noting. Intriguingly, ZLL is essential for maintaining stem cells of the shoot meristem in an undifferentiated state during the transition from embryo-specific development to repetitive organ formation through the self-perpetuating shoot meristem divisions (Moussian, 1998). AGO also plays an important role in maintaining normal apical shoot meristem function (Bohmert, 1998). Thus, the homology between piwi and ZLL and AGO further suggests the existence of a family of novel genes essential for stem cell division in diverse organisms (Cox, 1998).
Given the functional conservation of the piwi family genes between distant species such as Drosophila, C. elegans, and Arabidopsis, it is tempting to speculate that the piwi-mediated mechanism is also conserved in mammals and humans less distant from Drosophila. Given that C. elegans and Drosophila are separated by >1000 million years of evolution, and human and Arabidopsis are even further apart, this functional conservation could reflect the existence of a very ancient mechanism for stem cell maintenance and proliferation in a multicellular ancestor. The conserved piwi mechanism may mediate cell-cell interactions. In Drosophila, genetic mosaic and piwi expression analyses together suggest that piwi function is required in the apical nonmitotic somatic cells to control GSC division. Similarly, in A. thaliana, it is thought that ZLL is required to maintain the undifferentiated state of shoot meristem stem cells by relaying positional information, possibly by mediating cell-cell interactions within the center of the shoot meristem (Moussian, 1998). Further analysis of Piwi should shed light on this evolutionarily conserved cell-cell interaction mechanism (Cox, 1998).
In Drosophila, Piwi (P-element-induced wimpy testis), which encodes a protein of the Argonaute family, is essential for germ stem cell self-renewal. Piwi has recently been shown to be a nuclear protein involved in gene silencing of retrotransposons and controlling their mobilization in the male germline. However, little is known about the molecular mechanisms of Piwi-dependent gene silencing. This study shows that endogenous Piwi immunopurified from ovary specifically associates with small RNAs of 25-29 nucleotides in length. Piwi-associated small RNAs were identified by cloning and sequencing as repeat-associated small interfering RNAs (rasiRNAs) derived from repetitive regions, such as retrotransposon and heterochromatic regions, in the Drosophila genome. Northern blot analyses revealed that in vivo Piwi does not associate with microRNAs (miRNAs) and that guide siRNA was not loaded onto Piwi when siRNA duplex was added to ovary lysate. In vitro, recombinant Piwi exhibits target RNA cleavage activity. These data together imply that Piwi functions in nuclear RNA silencing as Slicer by associating specifically with rasiRNAs originating from repetitive targets (Saito, 2006).
To investigate the molecular function of Piwi in gene silencing, monoclonal antibodies were produced against the protein. As an antigen, the N-terminal region of Piwi was used that shows much less similarity to the other members of the fly Argonaute family. Western blotting using the Piwi-specific antibody revealed that Piwi is strongly expressed in the ovary and in the early embryo, but later in development the protein levels in the embryo decrease. Piwi was not detected in Schneider 2 (S2) cell lysate. To examine the expression pattern of Piwi in fly testis, ovary, and embryo, immunofluorescent staining was carried out. In ovarioles, Piwi was found to be clearly accumulated in nuclei of both GSCs and somatic cells such as terminal filament cells (TFCs), cap cells, and follicle cells. piwi mRNA is detected in TFCs, and myc-tagged Piwi protein is found in both somatic and germline cells in adult ovary. Relatively low expression was observed in cystoblasts, as in the case of myc-Piwi. In embryos, Piwi was found accumulated in nuclei of pole cells. In testis, Piwi appeared to be expressed in the hub, a tiny cluster of post-mitotic somatic cells localized at the apical tip of the testis and in cyst progenitor cells. The hub functions in the maintenance of GSC identity and influences its behavior, while Piwi is required for the asymmetric division of GSCs to produce and maintain a daughter GSC but is not essential for further differentiation of committed daughter cells. Piwi seems not to be expressed in male germline stem cells (GSCs). This observation agrees well with the Western data showing that the relative amount of Piwi in testis is quite low (Saito, 2006).
Immunoprecipitation was performed from an ovary lysate using the anti-Piwi antibody. Silver staining of the immunoprecipitate failed to reveal any prominent bands copurifying with Piwi under physiological conditions, although some weak bands were perceived. Western blotting confirmed that the discrete band of ~92 kDa was Piwi itself. Whether immunoprecipitated Piwi associates with any small RNAs was investigated. Total RNAs were isolated from the immunopurified Piwi complex, labeled with 32P-pCp, and separated on a denaturing acrylamide gel. As a control, the AGO1 complex—an essential factor in miRNA-mediated gene silencing—was also immunopurified from the ovary, and miRNAs associated with AGO1 were simultaneously visualized. The Piwi-associated small RNAs migrated slightly slower than miRNAs. Compared with the size markers, Piwi-associated small RNAs were estimated to be 25–29 nt in length. Northern blot analysis using probes specific for miRNAs (miR-1 and miR-310) clearly revealed that the Piwi-associated small RNAs are not miRNAs (Saito, 2006).
To identify the Piwi-associated small RNAs, they were cloned and sequenced. Of 800 clones sequenced, 392 hit to the Drosophila genomic sequences in databases. The largest class of cloned RNAs (84.1%) was rasiRNAs, originally found in a small RNA profiling study in Drosophila embryo and testis. Breakdown products of rRNA, tRNA, mRNA, and snoRNA were also obtained (6.6%, 0.2%, 4.8%, and 4.1%, respectively). It is noteworthy that no miRNAs were obtained in this experiment. Among the rasiRNAs (330 clones), ~40% (130 out of 330) matched to various kinds of transposable elements, including LTR (long terminal inverted repeat) retrotransposons and LINE (long interspersed nuclear elements)-like elements. Those LINE-like elements include Het-A and TART, both known to transpose only to the chromosome ends to maintain telomere length. Another 60% (200 out of 330) corresponded to heterochromatic regions in the genome. By looking at the nucleotide sequences of all the clones, it was noticed that the Piwi-associated rasiRNAs include both the sense and the anti-sense of a transposable transcript, although sense (+) orientation clones tended to be less abundant than those with an antisense (–) orientation, and that the first nucleotide at the 5' end was predominantly U (~78%) . In addition, the Piwi-associated rasiRNAs were cloned by the procedure originally developed for miRNA cloning, indicating that they contain 5' phosphates and 2',3'-hydroxyl termini. These characteristics suggested that the Piwi-associated rasiRNAs are produced from dsRNA precursors by an RNase III domain-containing enzyme. In some cases, such as stalker4, rasiRNAs are only found in an antisense (–) orientation. However, where the orientation bias of rasiRNA loading onto Piwi comes from is unknown and requires further investigation (Saito, 2006).
To confirm that rasiRNAs associated with Piwi indeed involve both the sense and antisense of a transcript, Northern blotting analysis was performed. roo rasiRNA was chosen for examination because it was one of most abundant clones obtained (11 out of 300) and in both sense and antisense orientations. Both sense and antisense roo rasiRNAs were detected in the immunopurified Piwi complex as expected. Neither was found in the AGO1 complex, indicating that the Piwi association with rasiRNAs is specific. roo rasiRNA that is derived from the roo antisense transcript (hereinafter, referred to it as roo anti-rasiRNA) seems more abundant in the Piwi complex than the roo rasiRNA that was derived from the roo transcript in the sense orientation. This was determined from the fact that the signal of roo anti-rasiRNA in the anti-Piwi lane is about threefold stronger than that of roo anti-oligo (10 fmol), whereas the signal of roo rasiRNA is a little less (~85%) compared with that of roo sense oligo (10 fmol). This observation correlated well with the fact that less roo rasiRNA than roo anti-rasiRNA was obtained in the cloning experiment. The Piwi–rasiRNA association was further verified by Northern blotting on total RNAs isolated from Piwi and AGO1 immunopurified complexes in ovary. rasiRNAs originating from copia and TART retrotransposon transcripts, and one of the repetitive elements located in the centromeric heterochromatin region, Responder, were detected only in Piwi but not in AGO1 complexes, demonstrating that the association with rasiRNAs is specific to Piwi. Whether Piwi was able to associate with siRNA was also investigated. luc siRNA duplex was first incubated in ovary lysate. Piwi and AGO2 complexes were then immunopurified from the lysate using specific antibodies to each protein. Northern blotting revealed that guide siRNA was specifically loaded onto AGO2 but not onto Piwi. These data strongly indicate the high specificity of Piwi association with rasiRNAs. It is suggested that a complex for loading rasiRNAs specifically onto Piwi exists; as the Dicer1/ Loquacious (R3D1) and Dicer2/R2D2 complexes load, after processing, mature miRNA and guide siRNA onto AGO1 and AGO2, respectively (Saito, 2006).
Using GST-tagged recombinant full-length AGO1 (GST-AGO1), it has been shown that AGO1 is able to cleave target RNA completely complementary to miRNA. In this study, full-length GST-Piwi was produced and the activity was examined in an in vitro target RNA cleavage assay. Interestingly, it was noticed that in the alignment of the Piwi domains of Piwi, AGO1, and AGO2, the D–D–H (Asp–Asp–His) motif, shown to be essential for Slicer activity of human Ago2, was not well conserved, and that the third residue was replaced with Lys. Thus, it was suspected that Piwi may not exhibit Slicer activity. However, it was observed that GST-Piwi was able to cleave luc target RNA when it was preincubated with luc guide siRNA (21 nt; luc 21) as well as GST-AGO1. By searching for peptide sequence similarity over species, it was noticed that the D–D–K triad is conserved in a member of the Argonautes in Drosophila pseudoobscura and one of the Piwi domain-containing proteins in Tetrahymena thermophila (Saito, 2006).
For several years, it has been thought that Piwi is involved in gene silencing of LTR retrotransposons since (1) Piwi is one of the members of the fly Argonaute family of proteins, and (2) piwi mutations cause higher expression levels of retrotransposon transcripts in vivo. Links between protein factors involved in RNAi and the silencing of endogenous transposable elements have also been made in other species like C. elegans and Chlamydomonas reinhardtii. However, evidence at molecular levels to support such a Piwi function has not hitherto been reported. This study is the first to show that Piwi is specifically associated with rasiRNAs in vivo and that Piwi has Slicer activity. The data suggest a novel, third kind of gene silencing pathway in Drosophila, following two distinct gene silencing pathways mediated by AGO1-miRNAs and AGO2-siRNAs. The existence of rasiRNAs in Drosophila has been demonstrated previously through investigation of small RNA expression profiling in testis and embryo. In that study, it was mentioned that rasiRNAs show some characteristics that suggest involvement of an RNase III domain enzyme in rasiRNA processing. In this study, it was observed that Piwi-associated rasiRNAs also show the same characteristics as those found in another study. Which protein factor(s) then produces rasiRNAs? Dicer1, Dicer2, and Drosha were not detected in Piwi complex immunopurified from ovary. Although it may be due to the epitope hindering by Piwi and processing factor association, it is possible that a yet-to-be unidentified protein(s) other than Dicer1, Dicer2, and Drosha might be the crutial factor(s) for the rasiRNA-producing process (Saito, 2006).
In fly testis, Piwi seems not to be expressed in GSCs, but its expression is clearly observed in the hub, the somatic cell cluster that functions to maintain GSC identity and influence its behavior. It is known that piwi1 causes male infertility due to severe defects in spermatogenesis; thus, the necessity of the Piwi function in the hub for such processes is quite apparent. Although it also will be important to determine if Piwi is associated with rasiRNAs in the testis, these findings have set a new stage for understanding how Piwi functions in the formation and maturation of GSCs in both ovary and testis (Saito, 2006).
Exons - 8
The Piwi protein is a highly basic (pI 9.6) novel protein with no obvious similarity to other known proteins or functional motifs in the databases. It is characterized by alternating basic and acidic regions and is particularly basic over the carboxy-terminal 100 amino acid residues. Hydropathy analysis indicates that the Piwi protein contains no significant local hydrophobic regions that could be potential signal peptide domains or transmembrane domains. Nuclear localization of the Piwi protein is predicted. The protein has 21 conserved protein kinase C phosphorylation sites, 14 casein kinase 2 phosphorylation sites, and 4 tyrosine phosphorylation sites, indicating its potential as a phosphorylation target (Cox, 1998).
To determine whether the Piwi protein is conserved during evolution, homologous sequences were sought at the protein level and two ORFs of unknown function were identified from C. elegans. An expressed sequence tag was identified from a human testis cDNA library. cDNAs for the two C. elegans genes were obtained, and they were given the names prg-1 and prg-2 (piwi-related gene) to verify their homology to Drosophila piwi. The prg-1 and prg-2 genes share 40.1% and 38.5% amino acid identity to piwi, respectively, over their entire length. In the carboxy-terminal 104 amino acid region, the homology increases to 55.8% and 56.7%, respectively. Moreover, prg-1 and prg-2 are 90% identical to each other over their full length and 98% identical at the carboxyl terminus. This high degree of homology suggests that prg-1 and prg-2 may represent a gene duplication event. The two clones differ primarily in that prg-1 is 60 amino acids longer at the amino terminus than prg-2. To isolate human piwi homologs, the human EST (0.9 kb) clone was sequenced and used to screen a human testis cDNA library. A resulting 2.3-kb partial cDNA, given the name hiwi (for human piwi), shows 47.1% identical amino acid sequence to the Drosophila piwi over its full length, with 58.7% identity at the carboxyl terminus. Interestingly, no piwi-related sequences were found from bacteria or yeast genomes whose entire sequences are known. This is consistent with the stem cell-related function of piwi and potentially of piwi-like genes specific for multicellular organisms (Cox, 1998).
In addition to the above piwi homologs, a large number of putative and known proteins from various animals and plants have been identified that share significant homology with piwi solely at their respective carboxyl termini. This indicates that piwi is a member of a large novel gene family, now termed the Argonaute family (Carmell, 2002). Within this family are 13 additional putative C. elegans proteins and three Arabidopsis thaliana proteins, two of which are required for meristem cell divisions (Bohmert, 1998 and Moussian, 1998). Particularly, between piwi and the three Arabidopsis genes [ZWILLE (ZLL), argonaute (ago), and argonaute-like], an overall homology of ~20% amino acid identity is observed. The homology increases to 32%-52% identity in four regions, 30-100 amino acid residues each, located throughout the length of the Piwi protein, including the highly conserved carboxy-terminal region. Given the role of ZLL and ago in meristem cell division, this homology may have important implications for a conserved stem cell mechanism. Piwi, PRG-1, PRG-2, and HIWI differ from ZLL and AGO proteins, and especially from the 13 additional putative C. elegans proteins, predominantly at the amino terminus, suggesting that this region may be involved in piwi-specific function. The carboxy-terminal conservation suggests that this region of Piwi may contain a novel functional domain that plays an important role for the general activity of these proteins in diverse biochemical processes, with the amino terminus rendering the specificity of the activity (Cox, 1998).
To examine the carboxy-terminal region of homology more closely, these sequences were aligned using Block Maker, a program designed to reveal characteristic regions of protein families. Block Maker analysis identifies a 43 amino acid domain conserved among all 22 proteins, within which 5 residues are absolutely conserved with defined spacing. Eight more residues are also conserved with defined spacing among all known genes across the phyla except for several C. elegans ORFs with unknown function. This region is referred to as the Piwi box and it is suggested that this protein signature represents a novel conserved functional motif. Piwi thus represents a novel class of evolutionarily conserved proteins with potentially conserved functions (Cox, 1998).
RNA silencing regulates gene expression through mRNA degradation, translation repression and chromatin remodelling. The fundamental engines of RNA silencing are RISC and RITS complexes, whose common components are 21-25 nt RNA and an Argonaute protein containing a PIWI domain of unknown function. The crystal structure of an archaeal Piwi protein (AfPiwi) is organised into two domains, one resembling the sugar-binding portion of the lac repressor and another with similarity to RNase H. Invariant residues and a coordinated metal ion lie in a pocket that surrounds the conserved C-terminus of the protein, defining a key functional region in the PIWI domain. Furthermore, two Asp residues, conserved in the majority of Argonaute sequences, align spatially with the catalytic Asp residues of RNase H-like catalytic sites, suggesting that in eukaryotic Argonaute proteins the RNase H-like domain may possess nuclease activity. The conserved region around the C-terminus of the PIWI domain, which is required for small interfering RNA (siRNA) binding to AfPiwi, may function as the receptor site for the obligatory 5' phosphate of siRNAs, thereby specifying the cleavage position of the target mRNA (Parker, 2004).
An allelic series of the novel argonaute mutant (ago1-1 to ago1-6) of the herbaceous plant Arabidopsis thaliana has been isolated. The ago1 mutation pleotropically affects general plant architecture. The apical shoot meristem generates rosette leaves and a single stem, but axillary meristems rarely develop. Rosette leaves lack a leaf blade but still show adaxial/abaxial differentiation. Instead of cauline leaves, filamentous structures without adaxial/abaxial differentiation develop along the stem and an abnormal inflorescence bearing infertile flowers with filamentous organs is produced. Two independent T-DNA insertions into the AGO1 locus led to the isolation of two corresponding genomic sequences as well as a complete cDNA. The AGO1 locus was mapped close to the marker mi291a on chromosome 1. Antisense expression of the cDNA results in a partial mutant phenotype. Sense expression causes some transgenic lines to develop goblet-like leaves and petals. The cDNA encodes a putative 115 kDa protein with sequence similarity to translation products of a novel gene family present in nematodes as well as humans. No specific function has been assigned to these genes. Similar proteins are not encoded by the genomes of yeast or bacteria, suggesting that AGO1 belongs to a novel class of genes with a function specific to multicellular organisms (Bohmert, 1998).
Postembryonic development in higher plants is marked by repetitive organ formation via a self-perpetuating stem cell system, the shoot meristem. Organs are initiated at the shoot meristem periphery, while a central zone harbors the stem cells. The ZWILLE (ZLL) gene is specifically required to establish the central-peripheral organization of the embryo apex and this step is critical for shoot meristem self-perpetuation. zll mutants correctly initiate expression of the shoot meristem-specific gene SHOOT MERISTEMLESS in early embryos, but fail to regulate its spatial expression pattern at later embryo stages and initiate differentiated structures in place of stem cells. The ZLL gene encodes a novel protein, and related sequences are highly conserved in multicellular plants and animals but are absent from bacteria and yeast. It is proposed that ZLL relays positional information required to maintain stem cells of the developing shoot meristem in an undifferentiated state during the transition from embryonic development to repetitive postembryonic organ formation (Moussian, 1998).
Several lines of evidence indicate that the adaxial leaf domain possesses a unique competence to form shoot apical meristems. Factors required for this competence are expected to cause a defect in shoot apical meristem formation when inactivated and to be expressed or active preferentially in the adaxial leaf domain. PINHEAD, a member of a family of proteins that includes the translation factor eIF2C, is required for reliable formation of primary and axillary shoot apical meristems. In addition to high-level expression in the vasculature, low-level PINHEAD expression defines a novel domain of positional identity in the plant. This domain consists of adaxial leaf primordia and the meristem. These findings suggest that the PINHEAD gene product may be a component of a hypothetical meristem forming competence factor. Defects are described in floral organ number and shape, as well as aberrant embryo and ovule development associated with pinhead mutants, thus elaborating on the role of PINHEAD in Arabidopsis development. In addition, embryos doubly mutant for PINHEAD and ARGONAUTE1, a related, ubiquitously expressed family member, fail to progress to bilateral symmetry and do not accumulate the SHOOT MERISTEMLESS protein. Therefore PINHEAD and ARGONAUTE1 together act to allow wild-type growth and gene expression patterns during embryogenesis (Lynn, 1999).
Introduction of transgene DNA may lead to specific degradation of RNAs that are homologous to the transgene transcribed sequence through phenomena named post-transcriptional gene silencing (PTGS) in plants, quelling in fungi, and RNA interference (RNAi) in animals. It was shown previously that PTGS, quelling, and RNAi require a set of related proteins (SGS2, QDE-1, and EGO-1, respectively). This study reports the isolation of Arabidopsis mutants impaired in PTGS which are affected at the Argonaute1 (AGO1) locus. AGO1 is similar to QDE-2 required for quelling and RDE-1 required for RNAi. Sequencing of ago1 mutants reveals one amino acid essential for PTGS that is also present in QDE-2 and RDE-1 in a highly conserved motif. Taken together, these results confirm the hypothesis that these processes derive from a common ancestral mechanism that controls expression of invading nucleic acid molecules at the post-transcriptional level. As opposed to rde-1 and qde-2 mutants, which are viable, ago1 mutants display several developmental abnormalities, including sterility. These results raise the possibility that PTGS, or at least some of its elements, could participate in the regulation of gene expression during development in plants (Fagard, 2000).
During development of the somatic macronucleus from the germline micronucleus in ciliates, chromosome rearrangements occur in which specific regions of DNA are eliminated and flanking regions are healed, either by religation or construction of telomeres. A gene, TWI1, has been identified in Tetrahymena thermophila that is homologous to piwi and is required for DNA elimination. Small RNAs are specifically expressed prior to chromosome rearrangement during conjugation. These RNAs are not observed in TWI1 knockout cells. The RNAs require PDD1, another gene required for rearrangement, for expression. It is proposed that these small RNAs function to specify sequences to be eliminated by a mechanism similar to RNA-mediated gene silencing (Mochizuki, 2002).
Like most ciliated protozoa, Tetrahymena exhibits nuclear dimorphism. Each cell contains a germline micronucleus and a somatic macronucleus. During the sexual process of conjugation, two cells pair and their micronuclei undergo meiosis, followed by fertilization. The zygotic nucleus divides to produce the next generation of macro- and micro-nuclei in each cell (Mochizuki, 2002).
Macro- and micro-nuclei differ in structure and function. During vegetative growth, the micronucleus is transcriptionally inert, while the macronucleus is transcriptionally active. The micronucleus is diploid and divides mitotically; the macronucleus is polyploid and divides by amitosis. Although derived from the same zygotic nucleus, they differ in the organization of their genomes. The macronucleus lacks ~15% of DNA sequences found in the zygotic nucleus and in the micronucleus. This is due to sequence elimination associated with two types of DNA rearrangements that occur in developing macronuclei during the late stages of conjugation. The first type occurs by deletion of ~6000 internal eliminated sequences (IESs), accompanied by ligation of the flanking macronucleus-destined sequences. IESs 0.5 to >20 kb in length occur in noncoding regions. Excision of IESs can occur reproducibly (within a few nucleotides) at a specific site, or (with similar fidelity) at a limited number of alternative sites. The second type of rearrangement involves chromosome breakage followed by small deletions (<50 bp) and addition of telomeres. This 'chromosome healing' occurs at precise locations, producing 2–300 macronuclear chromosomes from the 5 chromosomes in the micronuclear (haploid) genome. This type of eliminated sequence is referred to as a breakage eliminated sequence (BES). Little is known about the mechanisms that produce IESs or BESs. BESs contain a conserved, 15 bp chromosome breakage sequence (Cbs), a likely site for recognition by (unknown) proteins. The only common elements associated with IESs analyzed to date are short (<10 bp), direct repeats of varied sequences at the ends of the IESs that have no known function in IES removal. The flanking regions of some IESs have cis-acting sequences required for IES removal, but no sequence homology has been observed between different elements. Thus, how IESs are recognized and eliminated is obscure (Mochizuki, 2002).
DNA sequences in the parental macronucleus guide DNA rearrangements. In Tetrahymena, introduction of DNA containing an IES sequence into the parental macronucleus inhibits DNA elimination of that IES when the new macronucleus forms. In another ciliate, Paramecium, an IES in the G surface antigen coding region that had aberrantly been retained in the macronucleus inhibited elimination of this IES when the new macronucleus formed, and deletion of another (A type) surface antigen gene in macronuclei resulted in deletion of this locus in new macronuclei, even though the gene in the micronucleus was intact. These phenomena suggest that sequence-specific information is transferred from the parental to the new macronucleus (Mochizuki, 2002 and references therein).
Evidence is presented for involvement of small RNAs in DNA rearrangement in Tetrahymena, and it is proposed that these RNAs transfer sequence information from the old to the new macronucleus. This evidence derives from analysis of a gene related to piwi, a member of the PPD (PAZ and Piwi domains) gene family defined by conserved PAZ and Piwi domains of unknown function. Some PPD gene family members are involved in posttranscriptional (PTGS) and/or transcriptional (TGS) gene silencing evoked by introduction of transgenic DNA or RNA. For example, AGO1 is required for PTGS in Arabidopsis, qde-2 is required for PTGS (quelling) in Neurospora, and rde-1 is required for RNA interference (RNAi) in C. elegans. In Drosophila, AGO1 and AGO2 are involved in RNAi; aubergine (sting) is required for RNAi and for silencing the Stellate locus, and piwi is required for silencing induced by multiple copies of Adh. In many of these events, small (20-26 nt) RNAs, processed by a Dicer-related, double-stranded RNase, play a pivotal role, but how PPD genes are related to small RNAs is not well understood (Mochizuki, 2002).
The piwi-related gene TWI1 is required for DNA rearrangement in Tetrahymena thermophila. In TWI1 knockout cells, both IES elimination and chromosome breakage are affected. Small (~28 nt) RNAs specifically expressed during conjugation do not accumulate in the absence of TWI1 or PDD1 (another gene required for IES elimination) expression (Mochizuki, 2002).
It is hypothesized that micronuclear transcripts or the small RNAs derived from them (scan RNAs or scnRNAs) are then transferred to the new macronucleus, possibly in association with Twi1p, Pdd1p, and Pdd2p, all of which show the remarkable property of localizing to the new macronucleus after they appear in the old one. Interestingly, Pdd1p contains three chromodomains and Pdd3p (another protein associated with IESs during macronuclear development) contains one, and chromodomains can interact with RNA. In the new macronucleus, the selected scnRNAs are hypothesized to target the DNAs to which they are homologous for elimination. The scnRNAs (and proteins complexed with them) could participate in the elimination process directly, or change some state of the chromatin region with which they interact by chemical (DNA methylation or histone modifications) or structural (chromatin remodeling) modification. Enzymes required for IES excision could then recognize one of these 'marked' sequences and excise it (Mochizuki, 2002).
The high degree of sequence homology between piwi and its homologs in other organisms suggests a potential functional conservation. This hypothesis was tested in C. elegans. The injection of specific antisense RNA into the germ-line syncytium of C. elegans eliminates maternal and zygotic gene activity, producing a gene-specific loss-of-function effect that may persist through several generations. This technique, as refined by Fire (1998) and termed RNA-mediated interference (RNAi), was used to assess the function of prg-1 and prg-2, two C. elegans piwi homologs. Given the extremely high homology between prg-1 and prg-2, an anti-prg-1 RNA was used for injection to interfere with the function of both genes. The F1 progeny of the injected worms are designated prg-RNAi worms for simplicity (Cox, 1998).
In wild-type C. elegans, two germ-line precursor cells, Z2 and Z3, give rise to ~2000 germ cells in the adult hermaphrodite. Germ-line proliferation occurs throughout most of larval development (L1-L4) and continues in the adult. This proliferation and maintenance of the germ line requires signals from the somatic distal tip cell (DTC) at the tip of each gonadal arm. During gonadal development, DTC migration results in the formation of two U-shaped gonadal arms by the L4 stage. Germ-line proliferation is limited to the distal end of each arm, forming a mitotic proliferation zone (MPZ), which serves as the GSC equivalent in C. elegans. Moving proximally, near the U turn of the gonad, germ cells enter meiotic prophase and then further differentiate into gametes at the proximal half of each arm, producing sperm in L4 and then oocytes in young adults (Cox, 1998).
The phenotype of prg-RNAi worms was examined both by quantitating their fertility and by assessing their gonadal and germ-line development. The prg-RNAi worms show three major aspects of germ-line defects. (1) As indicated by the distribution of the number of progeny per F1 animal, the fertility difference between the prg-RNAi and control worms is highly significant. On average, prg-RNAi worms produced 92 progeny, with 75% of prg-RNAi worms giving rise to less than125 progeny. In contrast, control RNAi animals on average produced 191, with 90% of these animals giving rise to greater than125 progeny. The significant reduction of the fertility, as well as other defects described below, may still only reflect a partial loss of prg-1 and prg-2 function, because the RNAi technique is known to phenocopy partial rather than complete loss-of-function mutants (Cox, 1998).
(2) DNA staining reveals a dramatic shortening of the mitotic proliferation zone (MPZ) in prg-RNAi worms. On average, adult prg-RNAi worms exhibit a 50% reduction in the number of mitotic nuclei as compared with control animals. Associated with the reduction of the mitotic zone is a gonadal shortening. Fifty-seven percent of prg-RNAi worms exhibit a moderate to severe shortening. In the most severe case, the gonadal arm never makes the U turn. The number of sperm produced in these worms is greatly reduced as well. In contrast, only 7% of control animals exhibit a mild gonadal shortening. The above observed gonadal defects in prg-RNAi worms suggest that prg-1 and prg-2 are essential for germ-line proliferation and maintenance. Because the gonadal shortening may be due to a defect in DTC migration, it is also possible that prg-1 and prg-2 may play a role in proper gonadogenesis. (3) In addition to the MPZ shortening, the mitotic index in the remaining mitotic zone is further reduced by 5.5-fold in prg-RNAi worms with mildly to moderately shortened gonads. This indicates the important role of prg-1 and prg-2 in maintaining the mitotic ability of the germ-line nuclei (Cox, 1998).
RNAi is a gene-silencing phenomenon triggered by double-stranded (ds) RNA and involves the generation of 21 to 26 nt RNA segments that guide mRNA destruction. In Caenorhabditis elegans, lin-4 and let-7 encode small temporal RNAs (stRNAs) of 22 nt that regulate stage-specific development. Inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-1), and two homologs of rde-1 (alg-1 and alg-2), cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further dcr-1, alg-1, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7 stRNAs. These findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation (Grishok, 2001).
One of the remarkable aspects about RNA interference (RNAi) in C. elegans is that the trigger molecules, dsRNA, can be administered via the animal's food. Assays were performed to see whether this feature is a universal property of the species by testing numerous strains that have been isolated from different parts of the globe. Strains were assayed on bacterial clones that were genetically modified to produce dsRNA homologous to different C. elegans genes. All isolates, with one exception, were sensitive to RNAi by feeding dsRNA of a germline-expressed gene, par-1. The one exceptional isolate, from Hawaii, subsequently named PPW-1, had a defect in RNAi that was specific to the germline and was a result of multiple mutations in a PAZ/PIWI domain-containing protein. ppw-1 animals have a defect in RNAi specifically for those mRNA targets that are expressed in the germline: somatically expressed endogenous genes are fully sensitive. Also, when ppw-1 animals are compared to wild-type animals, transgenically driven GFP expression in somatic tissues is equally sensitive to RNAi. Interestingly, although ppw-1 animals are fully resistant to all clones inducing embryonic lethality, in some of these clones the hatched larvae fail to progress to adulthood, suggesting that RNAi is not completely lost. Deleting ppw-1 in the canonical C. elegans strain Bristol N2 makes it resistant to feeding of dsRNA directed against germline-expressed genes. PPW-1 is a member of the Argonaute family of proteins; this protein family acts in posttranscriptional gene silencing and development, and is homologous to the RNAi gene rde-1. These data indicate that at least two members of this family are required for complete and effective RNAi in C. elegans (Tijsterman, 2002).
The ppw-1 gene product is homologous to RDE-1. Because both proteins are required to trigger efficient RNAi in the germline upon environmentally administered dsRNA, it is suggested that both proteins play nonredundant roles in the RNAi reaction and perhaps stabilize the RNAi intermediates, i.e., siRNAs, at different steps in the reaction. A putative role for RDE-1 in stabilizing such intermediates comes from the observation that pos-1 siRNA species are not detected in rde-1 animals that were fed pos-1 dsRNA. These animals are, however, capable of siRNA production, because cell-free extracts prepared from rde-1 animals are capable of dicing dsRNA into siRNAs, and siRNA can be detected shortly (5 hr) after injection of radio-labeled dsRNA molecules into the gonad of rde-1 animals. This indicates that the Dicer reaction, by which dsRNA is diced into siRNAs, is not compromised by the absence of functional RDE-1, and that the absence of observable steady-state levels of these siRNAs is thus more likely a stability issue (Tijsterman, 2002).
Whether the Hawaiian strain, as a result of the defect in PPW-1, is more susceptible to invasive elements that threaten the genomic content, like DNA transposons or dsRNA viruses (at present, no viroid species are known to infect the nematode), is difficult to address, but it seems unlikely. (1) It is found that DNA transposition is still repressed in the germline of the Hawaiian strain, and (2) the genome of the Hawaiian strain is not loaded with more remnants of retroviruses, compared to the Bristol N2 strain. (3) Without apparent selection pressure in its natural habitat, multiple basepair changes accumulated in PPW-1 in the Hawaiian isolate without apparent loss of growth or fecundity (Tijsterman, 2002).
In summary, PPW-1, a member of the PIWI/RDE-1/Argonaute protein family, is involved in RNAi in the C. elegans germline, most noticeably when the trigger molecules, dsRNA, are environmentally provided, and one C. elegans isolate, which was obtained from Hawaiian soil, lacks this factor. The fact that loss of rde-1 as well as loss of ppw-1 results in an RNAi-defective phenotype (of which the defect in ppw-1 seems tissue specific) indicates that at least two members of this important protein family are required for complete and effective RNAi in C. elegans (Tijsterman, 2002).
Two genes have been identified, smedwi-1 and smedwi-2, expressed in the dividing adult stem cells (neoblasts) of the planarian Schmidtea mediterranea. Both genes encode proteins that belong to the Argonaute/PIWI protein family and that share highest homology with those proteins defined by Drosophila PIWI. RNA interference (RNAi) of smedwi-2 blocks regeneration, even though neoblasts are present, irradiation-sensitive, and capable of proliferating in response to wounding; smedwi-2(RNAi) neoblast progeny migrate to sites of cell turnover but, unlike normal cells, fail at replacing aged tissue. It is suggested that SMEDWI-2 functions within dividing neoblasts to support the generation of cells that promote regeneration and homeostasis. PIWI proteins may be universal regulators of the production of stem cell progeny competent for performing differentiated functions (Reddien, 2006).
Planarian regeneration, based upon totipotent stem cells, the neoblasts, provides a unique opportunity to study in vivo the molecular program that defines a stem cell. DjPiwi-1, a planarian homologue of Drosophila Piwi, is preferentially accumulated in small cells distributed along the midline of the dorsal parenchyma. DjPiwi-1 transcripts were not detectable after X-ray irradiation by whole mount in situ hybridization. Real time RTPCR analysis confirmed the significant reduction of DjPiwi-1 expression after X-ray treatment. However, the presence of residual DjPiwi-1 transcription suggests that, although the majority of DjPiwi-1-positive cells can be neoblasts, this gene is also expressed in differentiating/differentiated cells. During regeneration, DjPiwi-1-positive cells reorganize along the midline of the stump and no accumulation of hybridization signal was observed either in the blastema area or in the parenchymal region beneath the blastema. DjPiwi-1-positive cells, as well as the DjMCM2-expressing neoblasts located along the midline and those spread all over the parenchyma, showed a lower tolerance to X-ray with respect to the DjMCM2-expressing neoblasts distributed along the lateral lines of the parenchyma. Taken together, these findings suggest the presence of different neoblast subpopulations in planarians (Rossi, 2006).
Genes belonging to the piwi family are required for stem cell self-renewal in diverse organisms. Mouse homologs of piwi were cloned by RT-PCR using degenerative primers. The deduced amino acid sequences of mouse homologs MIWI and MILI show that each contains a well-conserved C-terminal PIWI domain and that each shares significant homology with PIWI and their human counterparts HIWI. Both miwi and mili are found in germ cells of adult testis by in situ hybridization, suggesting that these genes may function in spermatogenesis. Furthermore, mili was expressed in primordial germ cells (PGCs) of developing mouse embryos and may therefore play a role during germ cell formation. MIWI may be involved in RNA processing or translational regulation, since MIWI was found to possess RNA binding activity. The data suggest that miwi and mili regulate spermatogenesis and primordial germ cell production (Kuramochi-Miyagawa, 2001).
Subcellular localization analysis of Piwi shows that it is a nucleoplasmic protein. This implies two things: Piwi may be involved in post-transcriptional mRNA processing in the nucleus or, alternatively, Piwi may be involved in nuclear functions indirectly related to gene expression. The former possibility may be more likely, considering the functions of other members of the piwi/zwille family. Rabbit eukaryotic initiation factor 2C (eIF2C) is one of the family members. The protein fraction that includes eIFC2 stabilizes the ternary complex of Met-tRNA, GTP and eIF2 in the presence of mRNA. Recently, another new member, rde-1, was reported and is involved in RNA-mediated interference (RNAi). Still another family member, sting/aubergine of Drosophila is postulated to interfere with the 3' UTR of Oskar (Kuramochi-Miyagawa, 2001 and references therein).
Piwi is evidently localized in nucleus, but MILI and MIWI proteins are localized in cytoplasm. These data suggest that the proteins are involved in post-transcriptional regulation of mRNA in cytoplasm. Although circumstantial, these data raise the possibility that some of the family members may be involved in RNA post-transcriptional processing or translational regulation (Kuramochi-Miyagawa, 2001).
Based on the notion that MIWI or MILI may be involved in RNA processing, the RNA binding activity of these proteins was analyzed. Full length MIWI protein avidly binds to riboguanine polymers under high stringency and a fragment spanning amino acids between 541 and 678 is responsible for the binding. Cerutti (2000) advocates that Piwi family proteins commonly possess a Piwi domain that is a 300-amino-acid domain containing an ~40 amino-acid Piwi box in the middle of the domain. The N-terminus of the Piwi domain of MIWI is indispensable for its RNA binding activity. The region of C-terminus of Piwi domain is reported to be essential for Piwi function and its function might be the stabilization of the protein. Taken together, overall structure of Piwi domain would be necessary for the full function of MIWI. RNA binding activity of MILI could not be detected. Function may be different between MIWI and MILI, like their different expression patterns. However, the possibility of the RNA binding activity of MILI cannot be excluded. The sequence specificity of bound RNA and the function of MIWI in RNA processing and translational regulation remain open questions (Kuramochi-Miyagawa, 2001).
The piwi family genes, which are defined by conserved PAZ and Piwi domains, play important roles in stem cell self-renewal, RNA silencing, and translational regulation in various organisms. To reveal the function of the mammalian homolog of piwi, mice with targeted mutations in the Mili gene, which is one of three mouse homologs of piwi, were produced and analyzed. Spermatogenesis in the MILI-null mice is blocked completely at the early prophase of the first meiosis, from the zygotene to early pachytene, and the mice are sterile. However, primordial germ cell development and female germ cell production are not disturbed. Furthermore, MILI binds to MVH, an essential factor during the early spermatocyte stage. The similarities in the phenotypes of the MILI- and MVH-deficient mice and in the physical binding properties of MILI and MVH indicate a functional association of these proteins in post-transcriptional regulation. These data indicate that MILI is essential for the differentiation of spermatocytes (Kuramochi-Miyagawa, 2004).
The functions of the C. elegans homologs of piwi, prg-1 and prg-2, have been studied using RNA interference (RNAi); these genes are important for the mitotic ability of the germline nuclei and are essential for germline proliferation and maintenance. In this regard, the prgs are not only structural, but also functional homologs of piwi. It is intriguing to investigate whether the mammalian MILI and MIWI are functional homologs of Drosophila Piwi. A couple of lines of evidence suggest that the two mammalian structural homologs of Piwi, i.e., MILI and MIWI, inherit only a subset of piwi functions. One line of evidence comes from the analysis of gene targeting. In addition to its crucial roles in germline stem cells, Piwi has less-well characterized roles in early oogenesis and spermatogenesis, possibly including germline cyst mitosis, meiosis and egg chamber polarity. The expression of MIWI and MILI is restricted to germ lineages, and the gene-targeted animals show defective spermatogenesis. However, no defects of Mili-/- Miwi-/- mice are observed at the stage of testicular stem cells. Thus, Mili and Miwi may only represent a subset of piwi functions (Kuramochi-Miyagawa, 2004).
The other line of evidence is subcellular localization of MIWI and MILI. The Piwi protein can be localized either to the nucleoplasm in germline stem cells or in the cytoplasm co-localized with polar granules such as Vasa in early embryos. MILI and MIWI are found in the cytoplasm associated with MVH. This again reflects only a subset of the Piwi function. This function is also similar to Aubergine, another Drosophila Piwi family protein, that is recruited to the posterior pole in a vasa-dependent manner as a polar granule component. Interestingly, Aubergine remains exclusively in the cytoplasm even after pole cell formation. In addition, the levels of homology between MILI or MIWI and Aubergine (31.0% and 36.6%, respectively) are similar to those seen with Piwi (32.7% and 37.1%, respectively). Taking these data into consideration, it is conceivable that MILI and MIWI might be functionally more similar to Aubergine. Meanwhile, genome analysis has revealed the third mouse homolog of piwi and aubergine (Accession Number AY135692). An unanswered question remains: whether the third member will represent other functions of piwi and aubergine? (Kuramochi-Miyagawa, 2004).
The piwi family genes, defined by conserved PAZ and Piwi domains of unknown function, have been implicated in RNAi and related phenomena, such as post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) in several organisms. AGO1 (Argonaute) and QDE-2 are required for PTGS in Arabidopsis and Neurospora, respectively, and RDE-1 is required for RNAi in C. elegans. In Drosophila, mutations in piwi and aubergine block RNAi activation during egg maturation and perturb translational control during oogenesis. Aubergine has the ability to effect the silencing of Stellate, which is a tandemly repetitive gene. Furthermore, in Drosophila, AGO1 and AGO2 are involved in RNAi, and piwi is required for PTGS and for TGS, which is induced by multiple copies of Alcohol dehydrogenase. MILI and MIWI may be involved in similar silencing mechanisms required for spermatogenesis. The gene expression profiles between the control and the Mili-/- testes are being compared. This could give some clues about the function on gene silencing (Kuramochi-Miyagawa, 2004).
Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, C. elegans mutants resistant to dsRNA-mediated interference (RNAi) have been selected. Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. Implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing are discussed (Tabara, 1999).
RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, cleavage of injected dsRNA to produce the initial siRNA population is analyzed in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for the organism's viability or fertility. These results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduce the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs can circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed (Parrish, 2001).
Although translational regulation of maternal mRNA is important for proper development of the Drosophila embryo, few genes involved in this process have been identified. In this report, the role of aubergine in oskar translation is described. aubergine has been implicated in dorsoventral patterning, since eggs from aubergine mutant mothers are ventralized and seldom fertilized. Two new alleles of aubergine have been isolated in a novel genetic screen; it has been shown that aubergine is also required for posterior body patterning, since the small fraction of eggs from aubergine minus mothers that are fertilized develop into embryos that lack abdominal segmentation. Although aubergine mutations do not appear to affect the stability of either Oskar mRNA or protein, the level of Oskar protein is significantly reduced in aubergine mutants. Thus, aubergine is required to enhance Oskar translation. While aubergine-dependence is conferred upon Oskar mRNA by sequences in the Oskar 3' UTR, aubergine may influence Oskar translation through an interaction with sequences upstream of the Oskar 3' UTR (Wilson, 1996).
In Drosophila oocytes, activation of Oskar translation from a transcript localized to the posterior pole is an essential step in the organization of the pole plasm, specialized cytoplasm that contains germline and abdominal body patterning determinants. Oskar is a component of polar granules, large particles associated with the pole plasm and the germline precursor pole cells of the embryo. aubergine mutants fail to translate Oskar mRNA efficiently and are thus defective in posterior body patterning and pole cell formation. Aubergine protein is related to eukaryotic translation initiation factor 2C. Aubergine is recruited to the posterior pole in a vasa-dependent manner and is itself a polar granule component. Consistent with its presence in these structures, Aubergine is required for pole cell formation, independent of its initial role in Oskar translation. Unlike two other known polar granule components, Vasa and Oskar, Aubergine remains cytoplasmic after pole cell formation, suggesting that the roles of these proteins diverge during embryogenesis (Harris, 2001).
The sting mutation, caused by a P element inserted into polytene region 32D, was isolated by a screen for male sterile insertions in Drosophila melanogaster. This sterility is correlated with the presence of crystals in spermatocytes and spermatids that are structurally indistinguishable from those produced in males carrying a deficiency of the Y-linked crystal (cry) locus. In addition, their morphology is needle-like in Stellate (Ste) plus flies and star-shaped in Ste mutant flies, once again as observed in cry minus males. The sti mutation leads to meiotic drive of the sex chromosomes, and the strength of the phenomenon is correlated with the copy number of the repetitive Ste locus. The same correlation is also true for the penetrance of the male sterile mutation. A presumptive sti null allele results in male sterility and lethal maternal effect. The gene was cloned and shown to code for a putative protein that is 866 amino acids long. A C-terminal domain of 82 amino acids is identified that is well conserved in proteins from different organisms. The gene is expressed only in the germline of both sexes. The interaction of sting with the Ste locus can also be demonstrated at the molecular level. While an unprocessed 8-kb Ste primary transcript is expressed in wild-type males, in X/Y homozygous sti males, as in X/Y cry minus males, a 0.7-kb mRNA is produced (Schmidt, 1999).
Rabbit eIF2C (94kDa) has been shown to play important roles in the eukaryotic peptide chain initiation process. In this study, the primary structure of rabbit eIF2C is determined by cDNA cloning. A 3599-bp composite sequence, which contains a single open reading frame, translates into an 813-deduced amino acid sequence. Northern blot analysis of rabbit liver ploy(A)+ RNA yielded a single message species at approximately 4.6kb. Western blot analysis of rabbit reticulocyte lysate using polyclonal antibody against the 94kDa eIF2C detected a higher-molecular-weight polypeptide (140kDa). No 94kDa polypeptide was detected. Sequence analysis reveals that rabbit eIF2C has strong homology with a hypothetical protein in Caenorhabditis elegans (Zou, 1998).
A murine piwi gene (miwi) is essential for spermatogenesis. miwi encodes a cytoplasmic protein specifically expressed in spermatocytes and spermatids. miwi null mice display spermatogenic arrest at the beginning of the round spermatid stage, resembling the phenotype of CREM, a master regulator of spermiogenesis. Furthermore, mRNAs of ACT (activator of CREM in testis) and CREM target genes are downregulated in miwi null testes. Whereas MIWI and CREM do not regulate each other's expression, MIWI complexes with mRNAs of ACT and CREM target genes. Hence, MIWI may control spermiogenesis by regulating the stability of these mRNAs (Deng, 2002).
The miwi ORF predicts that the MIWI protein contains 862 amino acid residues, with a relative molecular mass (Mr) of 98,600 and an isoelectric point of 9.46. Except for a 100-200-amino acid stretch at the N terminus, MIWI shares significant homology over its entire length with other PIWI family proteins, such as MILI from mice, HIWI and HILI (41% identity) from humans, SEAWI from sea urchin (48% identity), PIWI (37% identity) and AUB (38% identity) from Drosophila, PRG-1 and PRG-2 from C. elegans (34% and 33% identity, respectively), and PAP from Paramecium (28% identity). Interestingly, most of these proteins are involved in the development of the germline or its equivalent. In addition, miwi shares significant homology to genes involved in RNA interference (rde-1 in C. elegans and qde-2 in Neurospora), meristem cell division (Zwille and Agonaute in Arabidopsis), and translational initiation (eIF2C1 and eIF2C2 in humans and GERp95 in rabbits). The homology is particularly high in the C-terminal PIWI domain and the 110-amino acid PAZ domain in the middle of this class of proteins, suggesting the potential importance of these regions for MIWI function (Deng, 2002).
How is MIWI involved in regulating spermiogenesis? Among the PIWI family proteins, AUB has been implicated in translational regulation in Drosophila. RDE-1 in C. elegans, QDE-2 in Neurospora, and eIF2C in mammalian systems are also involved in RNA-related processes. All PIWI family proteins share a highly conserved PIWI domain that is enriched with highly basic and highly charged residues. This region in MIWI can bind to ribohomopolymers in vitro and is essential for MIWI function. MIWI is found in ribonuclear protein complexes with mRNAs of ACT and CREM target genes. Thus, MIWI is likely an RNA binding protein that regulates its target RNAs by directly binding to them. The drastic reduction of ACE, RT7, TP1, MTEST82, and ACT mRNAs in the miwi null mutant further suggest that MIWI complexes with these mRNAs to ensure their stability. The stabilization of the ACT mRNA may be of particular importance, since ACT in turn activates the transcription of the postmeiotic genes. Interestingly, miwi null does not affect the expression of CREM proteins, suggesting that MIWI does not regulate postmeiotic genes by regulating CREM expression. The regulatory pathway proposed in this paper is likely to play a central role in spermiogenesis (Deng, 2002).
Nearly half of the mammalian genome is composed of repeated sequences. In Drosophila, Piwi proteins exert control over transposons. However, mammalian Piwi proteins, MIWI and MILI, partner with Piwi-interacting RNAs (piRNAs) that are depleted of repeat sequences, which raises questions about a role for mammalian Piwi's in transposon control. A search for murine small RNAs that might program Piwi proteins for transposon suppression revealed developmentally regulated piRNA loci, some of which resemble transposon master control loci of Drosophila. Evidence of an adaptive amplification loop is found in which MILI catalyzes the formation of piRNA 5' ends. Mili mutants derepress LINE-1 (L1) and intracisternal A particle and lose DNA methylation of L1 elements, demonstrating an evolutionarily conserved role for PIWI proteins in transposon suppression (Aravin, 2007a).
date revised: 15 April 2007
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