Tudor protein is expressed throughout the life cycle, beginning in the early stages of germ-line development in the female. During oogenesis, tud mRNA appears to be present in the oocyte precursor within the germarial cysts, and in stages 1-3 it accumulates within the developing oocyte. The transcript is localized to the posterior half of the oocyte during oogenetic stages 4-7 but is not detectable within the ooplasm by egg deposition and throughout early embryogenesis (Golumbeski, 1991).
Mutations in the tudor locus of Drosophila affect two distinct determinative processes in embryogenesis; segmentation of the abdomen and determination of the primordial germ cells. The distribution of Tudor protein during embryogenesis, and the effect of various mutations on its distribution, suggest that Tudor may carry out these functions separately, based on its location in the embryo. The protein is concentrated in the posterior pole cytoplasm (germ plasm), where it is found in polar granules and mitochondria. Throughout the rest of the embryo, Tudor is associated with the cleavage nuclei. Mutations in all maternal genes known to be required for the normal functioning of the germ plasm eliminate the posterior localization of Tudor, whereas mutations in genes required for the functioning of the abdominal determinant disrupt the localization around nuclei. Analysis of embryos of different maternal genotypes indicates that the average number of pole cells formed is correlated with the amount of Tudor protein that accumulates in the germ plasm. These results suggest that Tudor localized in the germ plasm is instrumental in germ cell determination, whereas nuclear-associated Tudor protein is involved in determination of segmental pattern in the abdomen (Bardsley, 1993).
Germ plasm, a specialized cytoplasm present at the posterior of the early Drosophila embryo, is necessary and sufficient for germ cell formation. Germ plasm is rich in mitochondria and contains electron dense structures called polar granules. To identify novel polar granule components, proteins were isolated that associate in early embryos with Vasa (Vas) and Tudor (Tud), two known polar granule associated molecules. Maternal expression at 31B (ME31B),eIF4A, Aubergine (AUB) and Transitional Endoplasmic Reticulum 94 (TER94) were identified as components of both Vas and Tud complexes and their localization to polar granules was confirmed by immuno-electron microscopy. ME31B, eIF4A and AUB are also present in processing (P) bodies, suggesting that polar granules, which are necessary for germ line formation, might be related to P bodies. The recovery of ER associated proteins TER94 and ME31B confirms that polar granules are closely linked to the translational machinery and to mRNP assembly (Thomson, 2008).
Little is understood of the molecular events that link the assembly of germ plasm to the formation of germ cells. There is a strong correlation between polar granule formation and germ cell formation, yet their functional relationship is still unclear. In an attempt to understand polar granule formation and function this study set out to isolate polar granule components with a biochemical approach; proteins common to both Tud and Vas complexes were isolated. These complexes were isolated by cross-linking proteins from early embryonic extracts followed by anti-Tud or anti-Vas immunoprecipitation; proteins found in both complexes were then immunolocalized using EM. Using this method it was confirmed that Aub is a polar granule component and three new polar granule components were identified: ME31B, TER94 and eIF4A. Through genetic interaction analysis in transheterozygous embryos it was shown that decreasing the levels of Vas or Tud along with either Aub, ME31B, Ter94 or eIF4A reduces germ cell number. This approach both identified novel polar granule components and implicated novel processes in germ cell formation (Thomson, 2008).
The presence of Aub, ME31B and eIF4A in polar granules supports the hypothesis that polar granules and P bodies are structurally, and perhaps functionally, related. Recovery of CUP, an ME31B-interacting protein, in the Tud complex further supports this. Similar parallels have recently been found for the mouse chromatoid body, an electron dense structure in the male germ line with similarities to Drosophila polar granules and nurse cell nuage. RNAs in P bodies are stored in a translationally quiescent state and can later be either degraded or translationally activated in response to physiological cues (Decker, 2006). Translational repression in P bodies occurs at the level of mRNA recruitment to the ribosome, and through miRNA silencing pathways. The polar granule components that were identified suggest their involvement in both types of post-transcriptional regulation. Aub has been implicated in processing of germ line specific piRNAs. Findings that Aub associates with polar granules implicates piRNAs in germ cell formation, as has a previous study. In contrast, Vas and eIF4A have been closely linked with translational regulation and are not known to participate in miRNA silencing pathways (Thomson, 2008).
The RNA-rich nature of early polar granules supports the idea that specific germ line-specific mRNAs are stored in polar granules in a translationally repressed state. Subsequently, these RNAs are translated and their function may be required for germ cell formation and further development. How could general translational repression mediated by polar granules be overcome? Conceivably Vas could be a key factor. Vas, a highly conserved polar granule component with homologues in other species involved in germ line formation, binds directly to eIF5B. Disrupting the eIF5B-Vas interaction abrogates germ cell formation, presumably due to the loss of the ability of Vas to initiate translation of yet unidentified mRNAs (Johnstone, 2004). Thus, Vas may act as a germ line specific mRNA translation derepression factor. Other tissue specific factors could adapt a P body to a specific function or cell line. Identification of mRNAs that localize in polar granules and are dependent on Vas for their translation will no doubt provide more insight into this mechanism (Thomson, 2008).
Ultrastructural analysis of proteins found in the Vas and Tud containing complexes revealed that polar granules were often in close proximity or in contact with ER. Supporting such a link, Ter94 and ME31B were present in both Tud and Vas complexes, and are enriched in polar granules. Further work is required to elucidate what proportion of polar granules associate with ER, and whether this association is stage dependent. The presence of Ter94, an ER exit site marker, with Vas, ME31B, Aub and eIF4A in the same structure suggests that ER exit sites directly associate with the translational machinery with both activating and repressing factors. Polar granules may form at ER exit sites, which could provide a mechanism for the localization and assembly of mRNPs required for the translational regulation of their constituent mRNAs. There is evidence that P bodies associate with ER exit sites. In the Drosophila ovary, Trailer Hitch (TRAL) associates with ER exit sites and associates with P body components such as ME31B and CUP. The C. elegans homologue of TRAL, Car-1, associates with DCAP-1, a P-body marker, and car-1 mutations affect ER assembly (Squirrell, 2006). A single TRAL peptide was recovered in one immunoprecipitation with Tud, perhaps lending additional support to an association between polar granules and ER exit sites (Thomson, 2008).
Repeated attempts to biochemically isolate polar granules were made over 30 years ago, before the advent of modern analytical techniques that allow the identification of very small amounts of protein. From this work a major polar granule component of approximately 95 kDa was identified. The nature of this protein was not determined although Ter94 has approximately the same molecular mass, as does PIWI, a 97-kDa likely polar granule component that has eluded the currently used screens. PIWI associates with Vas, a polar granule component, as well as with components of the miRNA machinery. PIWI RNA and protein are enriched in germ plasm and piwi mutants have defects in germ cell formation. The screen also did not identify Osk, which was shown by a yeast two-hybrid screen to bind directly to Vas. This may be because these proteins were not present in high enough abundance for detection. Alternatively, since the reactive ends of the cross-linkers that were used specifically cross-link cysteine residues, they would not stabilize a particular protein-protein interaction unless a pair of cysteine residues is within the range of the cross-linker. The work demonstrates that a molecular approach can be a powerful complement to genetics, and that purification schemes based on two independent reagents can reduce signal-to-noise problems that are inherent in co-immunoprecipitation experiments. Molecular approaches such as this one also have the capacity to identify proteins involved in a developmental process that are encoded by genes with multiple functions, or required for cellular viability, that will therefore elude phenotype-based genetic screens (Thomson, 2008).
Developmental analysis of a newly isolated maternal effect grandchildless mutant, tudor, in Drosophila indicates that tud+ activity is required during oogenesis for the determination and/or formation of primordial germ cells (pole cells) and for normal embryonic abdominal segmentation. Regardless of their genotype, progeny of females homozygous for strong alleles (tud1 and tud3) never form pole cells, apparently lack polar granules in the germ plasm, and approximately 40% of them die during late embryogenesis exhibiting severe abdominal segmentation pattern defects. Females carrying weak allele, tud4, produce progeny with some functional pole cells and form polar granules approximately one-third the size of those observed in wild-type oocytes and embryos. No segmentation abnormalities are observed in the inviable embryos derived from tud4/tud4 females (Boswell, 1985).
Pole cells and posterior segmentation in Drosophila are specified by maternally encoded genes whose products accumulate at the posterior pole of the oocyte. Among these genes is tudor. Progeny of hypomorphic tud mothers lack pole cells and have variable posterior patterning defects. A null allele was isolated to further investigate tud function. While no pole cells are ever observed in embryos from tud-null mothers, 15% of these embryos have normal posterior patterning. Oskar (Osk) and Vasa (Vas) proteins, and nanos (nos) RNA, all initially localize to the pole plasm of tud-null oocytes and embryos from tud-null mothers, while localization of germ cell-less (gcl) and polar granule component (pgc), is undetectable or severely reduced. In embryos from tud-null mothers, polar granules are greatly reduced in number, size, and electron density. Thus, tud is dispensable for somatic patterning, but essential for pole cell specification and polar granule formation (Thomson, 2004).
In Drosophila, polar plasm contains polar granules, which deposit the factors required for the formation of pole cells, germ line progenitors. Polar granules are tightly associated with mitochondria in early embryos, suggesting that mitochondria could contribute to pole cell formation. Mitochondrial large and small rRNAs (mtrRNAs) are transported from mitochondria to polar granules prior to pole cell formation and the large rRNA is essential for pole cell formation. The localization of mtrRNAs is diminished in embryos laid by tudor mutant females, although the polar granules are maintained. It was also found that Tud protein colocalizes with mtrRNAs at the boundaries between mitochondria and polar granules when the transport of mtrRNAs takes place. These observations suggest that Tud mediates the transport of mtrRNAs from mitochondria to polar granules (Amikura, 2001).
Mitochondria are organelles originated from a eubacterial symbiont and now functionally integrated into the eukaryotic cells. While the primary roles of the mitochondria are oxidative phosphorylation and biosynthesis of many metabolites, it has now become evident that they are also involved in the cellular events that play critical roles in development. One remarkable example is their involvement in germ line formation. In Drosophila, formation of the germ line progenitors, or pole cells, is induced by polar plasm localized in the posterior pole region of egg cytoplasm. The polar plasm contains mitochondria and polar granules which have been regarded as the structures essential for pole cell formation. Earlier ultrastructural studies have shown that both organelles become associated with each other at stages prior to pole cell formation, suggesting that mitochondria contribute to pole cell formation (Amikura, 2001).
Mitochondrial large rRNA (mtlrRNA) and small rRNA (mtsrRNA) are both transported from mitochondria and are localized on the surface of polar granules (hereafter the rRNAs outside mitochondria are referred to as extra-mitochondrial rRNAs). Injection of mtlrRNA restores pole-cell-forming ability to UV-irradiated embryos, and reduction of the extra-mitochondrial mtlrRNA results in the failure to form pole cells. These observations indicate that the extra-mitochondrial mtlrRNA on polar granules has an essential role in pole cell formation, presumably cooperating with mtsrRNA. Thus, the transport of mtrRNAs from mitochondria to polar granules is a critical step for pole cell formation. However, the molecule(s) mediating this transport remains elusive (Amikura, 2001).
It has been reported that the localization of the extra-mitochondrial mtrRNAs at the posterior pole region of embryos was impaired by mutation of any one of the maternally acting genes, namely oskar (osk), vasa (vas) and tudor (tud). These genes have been identified as the ones required for pole cell formation as well as for polar granule assembly. These genes all produce proteins localized to polar granules, and the association of these proteins with polar granules occurs stepwise and in a hierarchical manner: each subsequent localization depends on the activity of the preceding genes. The most downstream gene, tud, encodes a protein localized in mitochondria as well as in polar granules. This observation led to the idea that Tud may mediate the transport of mtrRNAs from mitochondria to polar granules (Amikura, 2001).
However, the possibility remained that the delocalization of extra-mitochondrial mtrRNAs from the posterior of tud mutant embryos is merely caused by their incapability to form polar granules, which tether the RNAs to the posterior. To exclude this possibility the effects of tud mutations on polar granules and mtrRNAs distribution were analyzed at a light and an electron microscopic level. In early embryos derived from tud mutant females, a polar granule component, Vas protein was normally localized in polar plasm, while extra-mitochondrial mtrRNAs were undetectable in the cytoplasm. Compatible with this observation, tud mutant embryos contained polar granules, although their number and size were decreased. These polar granules were associated with mitochondria during the early cleavage stage. However, no mtrRNA signal was observed in the polar granules. In normal embryos, Tud and mtrRNAs colocalized at the boundaries between mitochondria and polar granules, when the transport of mtrRNAs takes place. These ultrastructural data strongly support the idea that Tud mediates the transport of mtrRNAs from mitochondria to polar granules (Amikura, 2001).
Search PubMed for articles about Drosophila Tudor
Amikura, R., Hanyu, K., Kashikawa, M. and Kobayashi, S. (2001). Tudor protein is essential for the localization of mitochondrial RNAs in polar granules of Drosophila embryos. Mech. Dev. 107: 97-104. Medline abstract: 11520666
Ancelin, K., Lange, U. C., Hajkova, P., Schneider, R., Bannister, A. J., Kouzarides, T. and Surani, M. A. (2006). Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse germ cells. Nat. Cell Biol. 8: 623-630. Medline abstract: 16699504
Anne, J. and Mechler, B. M. (2005). Valois, a component of the nuage and pole plasm, is involved in assembly of these structures, and binds to Tudor and the methyltransferase Capsuleen. Development 132(9): 2167-77. 15800004
Anne, J. et al. (2007). Arginine methyltransferase Capsuléen is essential for methylation of spliceosomal Sm proteins and germ cell formation in Drosophila. Development 134: 137-146. Medline abstract: 17164419
Arkov, A. L., Wang, J.-Y. S., Ramos, A. and Lehmann, R. (2006). The role of Tudor domains in germline development and polar granule architecture. Development 133: 4053-4062. Medline abstract: 16971472
Bardsley, A., McDonald, K. and Boswell, R. E. (1993). Distribution of Tudor protein in the Drosophila embryo suggests separation of functions based on site of localization. Development 119: 207-219. 8275857
Boswell, R. E. and Mahowald, A. P. (1985). tudor, a gene required for assembly of the germ plasm in Drosophila melanogaster. Cell 43: 97-104. Medline abstract: 3935320
Brahms, H., Meheus, L., de Brabandere, V., Fischer, U. and Lührmann, R. (2001). Symmetrical dimethylation of arginine residues in spliceosomal Sm protein B/B' and the Sm-like protein LSm4, and their interaction with the SMN protein. RNA 7: 1531-1542. Medline abstract: 11720283
Bühler, D., Raker, V., Lührmann, R. and Fischer, U. (1999). Essential role for the tudor domain of SMN in spliceosomal U snRNP assembly: implications for spinal muscular atrophy. Hum. Mol. Genet. 8: 2351-2357. Medline abstract: 10556282
Cavey, M., Hijal, S., Zhang, X. and Suter, B. (2005). Drosophila valois encodes a divergent WD protein that is required for Vasa localization and Oskar protein accumulation. Development 132(3): 459-68. 15634703
Charier, G., Couprie, J., Alpha-Bazin, B., Meyer, V., Quéméneur, E., Guérois, R., Callebaut, I., Gilquin, B. and Zinn-Justin, S. (2004). The Tudor tandem of 53BP1: a new structural motif involved in DNA and RG-rich peptide binding. Structure 12: 1551-1562. Medline abstract: 15341721
Cheng, D., Cote, J., Shaaban, S. and Bedford, M. T. (2007). The arginine methyltransferase CARM1 regulates the coupling of transcription and mRNA processing. Mol. Cell 25(1): 71-83. Medline abstract: 17218272
Côté, J. and Richard, S. (2005). Tudor domains bind symmetrical dimethylated arginines. J. Biol. Chem. 280: 28476-28483. Medline abstract: 15955813
Chuma, S., et al. (2003). Mouse Tudor Repeat-1 (MTR-1) is a novel component of chromatoid bodies/nuages in male germ cells and forms a complex with snRNPs. Mech. Dev. 120(9): 979-90. Medline abstract: 14550528
Chuma, S., et al. (2006). Tdrd1/Mtr-1, a tudor-related gene, is essential for male germ-cell differentiation and nuage/germinal granule formation in mice. Proc. Natl. Acad. Sci. 103(43): 15894-9. Medline abstract: 17038506
Decker, C. J. and Parker, R. (2006). CAR-1 and trailer hitch: driving mRNP granule function at the ER. J. Cell Biol. 173: 159-163. PubMed Citation: 16636142
Ephrussi, A. and Lehmann, R. (1992). Induction of germ cell formation by oskar. Nature 358: 387-392. Medline abstract: 1641021
Gavis, E. R. and Lehmann, R. (1994). Translational regulation of nanos by RNA localization. Nature 369: 315-318. Medline abstract: 7514276
Golumbeski, G. S., Bardsley, A., Tax, F. and Boswell, R. E. (1991). tudor, a posterior-group gene of Drosophila melanogaster, encodes a novel protein and an mRNA localized during mid-oogenesis. Genes Dev. 5: 2060-2070. Medline abstract: 1936993
Gonsalvez, G. B., Rajendra, T. K., Tian, L. and Matera, A. G. (2006). The Sm-protein methyl transferase, dart5, is essential for germ-cell specification and maintenance. Curr. Biol. 16: 1077-1089. Medline abstract: 16753561
Hay, B., Jan, L. Y. and Jan, Y. N. (1990). Localization of vasa, a component of Drosophila polar granules, in maternal-effect mutants that alter embryonic anteroposterior polarity. Development 109: 425-33
Hosokawa, M., et al. (2007). Tudor-related proteins TDRD1/MTR-1, TDRD6 and TDRD7/TRAP: domain composition, intracellular localization, and function in male germ cells in mice. Dev. Biol. 301(1): 38-52. Medline abstract: 17141210
Huang, Y., Fang, J., Bedford, M. T., Zhang, Y. and Xu, R. M. (2006). Recognition of histone H3 lysine-4 methylation by the double tudor domain of JMJD2A. Science 312:748-751. Medline abstract: 16601153
Huyen, Y., Zgheib, O., Ditullio, R. A., Jr, Gorgoulis, V. G., Zacharatos, P., Petty, T. J., Sheston, E. A., Mellert, H. S., Stavridi, E. S. and Halazonetis, T. D. (2004). Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks. Nature 432: 406-411. Medline abstract: 15525939
Johnstone, O. and Lasko, P. (2004). Interaction with eIF5B is essential for Vasa function during development. Development 131: 4167-4178. 15280213
Kim, J., Daniel, J., Espejo, A., Lake, A., Krishna, M., Xia, L., Zhang, Y. and Bedford, M. T. (2006). Tudor, MBT and chromo domains gauge the degree of lysine methylation. EMBO Rep. 7: 397-403. Medline abstract: 16415788
Kotani, T., et al. (2007). A novel mutation at the N-terminal of SMN Tudor domain inhibits its interaction with target proteins. J. Neurol. 254(5): 624-30. Medline abstract: 17415510
Lasko, P. F. and Ashburner, M. (1990). Posterior localization of vasa protein correlates with, but is not sufficient for, pole cell development. Genes Dev. 4(6): 905-21. PubMed citation: 2384213
Maurer-Stroh, S., Dickens, N. J., Hughes-Davies, L., Kouzarides, T., Eisenhaber, F. and Ponting, C. P. (2003). The Tudor domain 'Royal Family': Tudor, plant Agenet, Chromo, PWWP and MBT domains. Trends Biochem. Sci. 28: 69-74. Medline abstract: 12575993
Nakamura, A., Amikura, R., Mukai, M., Kobayashi, S. and Lasko, P. F. (1996). Requirement for a noncoding RNA in Drosophila polar granules for germ cell establishment. Science 274: 2075-2079. Medline abstract: 8953037
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Squirrell, J. M., et al. (2006). CAR-1, a protein that localizes with the mRNA decapping component DCAP-1, is required for cytokinesis and ER organization in Caenorhabditis elegans embryos. Mol. Biol. Cell 17: 336-344. PubMed Citation: 1626726
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Thomson, T. and Lasko, P. (2004). Drosophila tudor is essential for polar granule assembly and pole cell specification, but not for posterior patterning. Genesis 40: 164-170. Medline abstract: 15495201
Thomson, T. and Lasko, P. (2005). Tudor and its domains: germ cell formation from a Tudor perspective. Cell Res. 15: 281-291. Medline abstract: 15857583
Thomson, T., Liu, N., Arkov, A., Lehmann, R. and Lasko, P. (2008). Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins. Mech. Dev. 125(9-10): 865-73. PubMed Citation:
Vo, H. D. L., Wahiduzzaman, Tindell, S. J., Zheng, J., Gao, M. and Arkov, A. L. (2019). Protein components of ribonucleoprotein granules from Drosophila germ cells oligomerize and show distinct spatial organization during germline development. Sci Rep 9(1): 19190. PubMed ID: 31844131
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date revised: 15 April 2020
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