Vasa is an RNA binding protein with an ATP-dependent RNA helicase that is a member of the DEAD box family of proteins. The vasa gene is essential for germ cell development and was first identified in Drosophila melanogaster,[1] but has since been found to be conserved in a variety of vertebrates and invertebrates including humans.[2][3] The Vasa protein is found primarily in germ cells in embryos and adults,[2] where it is involved in germ cell determination and function, as well as in multipotent stem cells, where its exact function is unknown.[3]

Gene

The Vasa gene is a member of the DEAD box family of RNA helicases in Drosophila melanogaster.[1] Its human ortholog, Ddx4, is located on human chromosome 5q. It is syntenic to mouse chromosome 13, where the mouse vasa gene is located.[2] The gene is conserved in many invertebrates and vertebrate species such as Caenorhabditis elegans, Xenopus, Zebrafish, flatworms, echinoderms, molluscs, nematodes, mice and rats as an important part of germ line maintenance and function.[2][3]

All vertebrate species and Drosophila have only one vasa ortholog. However, C. elegans has four Vasa genes, of which only one (GLH-1) is essential.[4][5]

All DEAD box genes, including Vasa, have 9 conserved sequence motifs.[6] The Vasa gene family evolved from a duplication event followed by acquiring certain domains.[7] Early in the evolution of multicellular animals, the duplication of PL10 related DEAD-box gene occurred.[8] This resulted in animals having both Vasa and PL10 genes, but plants and fungi only have PL10 genes and no Vasa.[7] After the duplication event, the N-terminal region acquired Zn-knuckle domains which are now conserved in invertebrates. Vertebrates and insects both have lost the Zn-knuckle domains. The number of these domains vary between different species Vasa genes. An important property of Zn-knuckles, which can be categorized as classical zinc fingers,[9] is that they are able to bind to single and double stranded DNA or RNA.[10] The presence of Zn-knuckles in invertebrates and absence in vertebrates may be an indication of differences in target binding sites. Their presence may be important to functions outside germ line development. An exception to this theory is the presence of Zn-knuckles in all four C. elegans Vasa genes, which are restricted to functions in the germ line. [11]

Protein

The protein product in humans has 724 amino acids, a molecular mass of 79 kDa and 8 conserved domains in all DEAD-box proteins that is involved in RNA helicase activity. Domain V contains the DEAD motif.[12] As with other Vasa related proteins, human Vasa has a N terminus rich in glycine and RGG motif repeats that function in RNA binding.[13]

Vasa is regulated at the transcript and protein level. Developing embryos and adults regulate Vasa expression to cell and tissue specific locations. In Drosophila, zygotic transcription of Vasa occurs at pole cells, and stays germ-line specific throughout the life of the organism.[14]

The Vasa promoter is regulated through methylation. In cells were Vasa is transcribed successfully, the promoter is hypomethylated and in all other cells it is methylated.[14] When Vasa is hypermethylated in testes, spermatogenesis defects may occur.[15]

Post-transcriptionally Vasa has several splice forms in different animals.[16][17] In Parhyale hawaiensis, Vasa transcript is uniformly distributed in the embryo and is localized depending on the stabilization of the 3’UTR (Untranslated Region to the germ line cells.[18] Translation can be inhibited by cis regulatory elements in the transcript's 5' and 3' UTRs. They may inhibit translation by forming secondary RNA structures or binding trans-acting factors. Vasa expression localization is directed by repressing these translation inhibitory pathways.[19]

Post-translationally, in Drosophila, Vasa protein is localized to the pole plasm during embryonic development. Many other proteins in Drosophila are also localized to the poles. For example, oskar protein was found to localize to pole plasm and may be involved in anchoring Vasa to polar granules in the posterior pole of the oocyte.[20] Another enzyme, fat facets, may further stabilize Vasa in the pole plasm.[21] Other post-translational modification includes phosphorylation of the Vasa ortholog in C. elegans,[22] and arginine methylation in a conserved region of mice, Xenopus and Drosophila Vasa genes. [23]

Function

One of main function of Vasa protein is in germ cell determination and function.[2] It uses ATP dependent RNA helicase catalytic activity to regulate the translation of multiple mRNAs.[24] Vasa unwinds the duplex RNA by binding and bending short stretches of the duplex in a non-processive manner.[25][26] The conserved domain may act as chaperones by unwinding RNA secondary structures and refolding properly.[27] pre-mRNA splicing, ribosome biogenesis, nuclear export, translational regulation and degradation.[6]

Vasa was found to bind RNA in a sequence-specific manner. In the Drosophila embryos, Vasa binds the Uracil rich motif of the mei-P26 UTR. A mutation in Vasa reduced the interaction of between Mei-P26 and initiation factor elF58 which in turn significantly reduced translation of the gene.[28]

Recent evidence in invertebrates suggests that Vasa has a role in multipotent stem cells, but the exact function is unknown.[3]

Mutations

Drosophila

A null mutation causes female sterility due to severe defects in oogenesis[29] but males are fertile.

Homozygous mutations for partial loss of function allows eggs to be fertilized but embryos lack germ cells.[8]

Mus musculus

Mutations in Vasa homolog, Mvh, cause defects in spermatogenesis but females are fertile. Male sterility may be due to deficiencies in germ cell proliferation and differentiation (the mouse homolog of Droso.). Female fertility may be due to functional redundancy by other DEAD-box family members. Null mutation still allows primordial germ cells to form but have severe defects.[30]

Homo sapiens

Although there are no studies done on Vasa mutations in humans, it is likely that it would cause sterility.[2]

These sex-specific phenotypes in mice and Drosophila mutants suggest that Vasa either regulated differently or has different target functions in the two germ line types.[3]

Tissue, and subcellular distribution

Vasa expression is restricted to tissue specific cells. Until recently it was thought that Vasa protein can only be found in gametes and is undetectable in somatic cells.[2] Within germ cells, Vasa is expressed in the cytoplasm. During embryogenesis, Vasa is expressed in migratory primordial germ cells (PGCs) at the gonadal ridge in both males and females. This specificity allows Vasa to be used as a highly specific marker for germ cells.[2] In a patient with Sertoli cell syndrome, no Vasa signal was detected from testicular biopsy.[2] However, recent studies show that Vasa functions in other cells as well.[3]

A study on Macrostomum lignano found Vasa expression in multipotent neoblast stem cells in addition to germ cells.[16] However, RNAi knockdown revealed that either Vasa is non-essential in this organism or is made functionally redundant by other Vasa-like genes. Similar results were found on studies of the colonial ascidian Botryllus primigenus,[31] oysters,[32] teleosts,[33] clawed frog,[34] the parasitic wasp,[35] and the crustacean Parhyale hawaiensis.[18]

Vasa expression has been observed in epithelial ovarian cancer cells. It was found to deter the DNA damage-induced G2 checkpoint by downregulating the expression of another gene.[36] Vasa is also present in chicken embryonic stem cells where it induces expression of germ line genes. This function still supports the most important role of Vasa in germ line development.[37] In Cnidarians, Vasa has a role in nerve cells and gland cells.[38] Other examples include Vasa in multipotent stem cell cluster of Polyascus polygenea buds and stolon,[39] Vasa in auxiliary cells of oyster ovaries,[40] Vasa in non-germ-line lineages in the snail Ilyanassa,[41] Vasa in progenitor mesodermal posterior growth zone of the polychaete annelid Platynereis dumerilii,[17] and Vasa present in non-genetical segments during Oligochaete development.[42] But no reports of vasa expressed outside of germ line cells in vertebrates or insects.[3]

Expression

In Drosophila, vasa expression is seen in germ cells, specifically the germline stem cells (GSC's) of female ovaries and in the early stages of spermatogenesis in the male testis.

Staining

Due to the localization of vasa, immunohistochemistry staining can be done with vasa antibodies. For example, vasa antibody staining is specific for germ cells in the D. melanogaster germarium.

This protein is localized to the cytoplasm of fetal germ cells and to the cytoplasm of developing oocytes in mammals.

References

  1. 1 2 Raz E (2000). "The function and regulation of vasa-like genes in germ-cell development". Genome Biology. 1 (3): REVIEWS1017. doi:10.1186/gb-2000-1-3-reviews1017. PMC 138859. PMID 11178242.
  2. 1 2 3 4 5 6 7 8 9 Castrillon DH, Quade BJ, Wang TY, Quigley C, Crum CP (August 2000). "The human VASA gene is specifically expressed in the germ cell lineage". Proceedings of the National Academy of Sciences of the United States of America. 97 (17): 9585–90. Bibcode:2000PNAS...97.9585C. doi:10.1073/pnas.160274797. PMC 16908. PMID 10920202.
  3. 1 2 3 4 5 6 7 Gustafson EA, Wessel GM (July 2010). "Vasa genes: emerging roles in the germ line and in multipotent cells". BioEssays. 32 (7): 626–37. doi:10.1002/bies.201000001. PMC 3090673. PMID 20586054.
  4. ↑ Kuznicki KA, Smith PA, Leung-Chiu WM, Estevez AO, Scott HC, Bennett KL (July 2000). "Combinatorial RNA interference indicates GLH-4 can compensate for GLH-1; these two P granule components are critical for fertility in C. elegans". Development. 127 (13): 2907–16. doi:10.1242/dev.127.13.2907. PMID 10851135.
  5. ↑ Spike C, Meyer N, Racen E, Orsborn A, Kirchner J, Kuznicki K, Yee C, Bennett K, Strome S (April 2008). "Genetic analysis of the Caenorhabditis elegans GLH family of P-granule proteins". Genetics. 178 (4): 1973–87. doi:10.1534/genetics.107.083469. PMC 2323790. PMID 18430929.
  6. 1 2 Linder P (2006). "Dead-box proteins: a family affair--active and passive players in RNP-remodeling". Nucleic Acids Research. 34 (15): 4168–80. doi:10.1093/nar/gkl468. PMC 1616962. PMID 16936318.
  7. 1 2 Mochizuki K, Nishimiya-Fujisawa C, Fujisawa T (June 2001). "Universal occurrence of the vasa-related genes among metazoans and their germline expression in Hydra". Development Genes and Evolution. 211 (6): 299–308. doi:10.1007/s004270100156. PMID 11466525. S2CID 20585574.
  8. 1 2 Schüpbach T, Wieschaus E (July 1986). "Maternal-effect mutations altering the anterior-posterior pattern of the Drosophila embryo". Roux's Archives of Developmental Biology. 195 (5): 302–317. doi:10.1007/bf00376063. PMID 28306055. S2CID 274353.
  9. ↑ Gamsjaeger R, Liew CK, Loughlin FE, Crossley M, Mackay JP (February 2007). "Sticky fingers: zinc-fingers as protein-recognition motifs". Trends in Biochemical Sciences. 32 (2): 63–70. doi:10.1016/j.tibs.2006.12.007. PMID 17210253.
  10. ↑ Rajavashisth TB, Taylor AK, Andalibi A, Svenson KL, Lusis AJ (August 1989). "Identification of a zinc finger protein that binds to the sterol regulatory element". Science. 245 (4918): 640–3. Bibcode:1989Sci...245..640R. doi:10.1126/science.2562787. PMID 2562787.
  11. ↑ Gruidl ME, Smith PA, Kuznicki KA, McCrone JS, Kirchner J, Roussell DL, Strome S, Bennett KL (November 1996). "Multiple potential germ-line helicases are components of the germ-line-specific P granules of Caenorhabditis elegans". Proceedings of the National Academy of Sciences of the United States of America. 93 (24): 13837–42. Bibcode:1996PNAS...9313837G. doi:10.1073/pnas.93.24.13837. PMC 19442. PMID 8943022.
  12. ↑ Castrillon DH, Quade BJ, Wang TY, Quigley C, Crum CP (August 2000). "The human VASA gene is specifically expressed in the germ cell lineage". Proceedings of the National Academy of Sciences of the United States of America. 97 (17): 9585–90. Bibcode:2000PNAS...97.9585C. doi:10.1073/pnas.160274797. PMC 16908. PMID 10920202.
  13. ↑ Lüking A, Stahl U, Schmidt U (1998). "The protein family of RNA helicases". Critical Reviews in Biochemistry and Molecular Biology. 33 (4): 259–96. doi:10.1080/10409239891204233. PMID 9747670.
  14. 1 2 Kitamura E, Igarashi J, Morohashi A, Hida N, Oinuma T, Nemoto N, Song F, Ghosh S, Held WA, Yoshida-Noro C, Nagase H (March 2007). "Analysis of tissue-specific differentially methylated regions (TDMs) in humans". Genomics. 89 (3): 326–37. doi:10.1016/j.ygeno.2006.11.006. PMC 1847344. PMID 17188838.
  15. ↑ Sugimoto K, Koh E, Sin HS, Maeda Y, Narimoto K, Izumi K, Kobori Y, Kitamura E, Nagase H, Yoshida A, Namiki M (August 2009). "Tissue-specific differentially methylated regions of the human VASA gene are potentially associated with maturation arrest phenotype in the testis". Journal of Human Genetics. 54 (8): 450–6. doi:10.1038/jhg.2009.59. PMID 19629140.
  16. 1 2 Pfister D, De Mulder K, Hartenstein V, Kuales G, Borgonie G, Marx F, Morris J, Ladurner P (July 2008). "Flatworm stem cells and the germ line: developmental and evolutionary implications of macvasa expression in Macrostomum lignano". Developmental Biology. 319 (1): 146–59. doi:10.1016/j.ydbio.2008.02.045. PMID 18405892.
  17. 1 2 Rebscher N, Zelada-González F, Banisch TU, Raible F, Arendt D (June 2007). "Vasa unveils a common origin of germ cells and of somatic stem cells from the posterior growth zone in the polychaete Platynereis dumerilii". Developmental Biology. 306 (2): 599–611. doi:10.1016/j.ydbio.2007.03.521. PMID 17467683.
  18. 1 2 Ozhan-Kizil G, Havemann J, Gerberding M (March 2009). "Germ cells in the crustacean Parhyale hawaiensis depend on Vasa protein for their maintenance but not for their formation". Developmental Biology. 327 (1): 230–9. doi:10.1016/j.ydbio.2008.10.028. PMID 19013453.
  19. ↑ Chatterjee S, Pal JK (May 2009). "Role of 5'- and 3'-untranslated regions of mRNAs in human diseases". Biology of the Cell. 101 (5): 251–62. doi:10.1042/BC20080104. PMID 19275763.
  20. ↑ Breitwieser W, Markussen FH, Horstmann H, Ephrussi A (September 1996). "Oskar protein interaction with Vasa represents an essential step in polar granule assembly". Genes & Development. 10 (17): 2179–88. doi:10.1101/gad.10.17.2179. PMID 8804312.
  21. ↑ Liu N, Dansereau DA, Lasko P (October 2003). "Fat facets interacts with vasa in the Drosophila pole plasm and protects it from degradation". Current Biology. 13 (21): 1905–9. doi:10.1016/j.cub.2003.10.026. PMID 14588248.
  22. ↑ Orsborn AM, Li W, McEwen TJ, Mizuno T, Kuzmin E, Matsumoto K, Bennett KL (September 2007). "GLH-1, the C. elegans P granule protein, is controlled by the JNK KGB-1 and by the COP9 subunit CSN-5". Development. 134 (18): 3383–92. doi:10.1242/dev.005181. PMID 17699606.
  23. ↑ Kirino Y, Vourekas A, Kim N, de Lima Alves F, Rappsilber J, Klein PS, Jongens TA, Mourelatos Z (March 2010). "Arginine methylation of vasa protein is conserved across phyla". The Journal of Biological Chemistry. 285 (11): 8148–54. doi:10.1074/jbc.M109.089821. PMC 2832966. PMID 20080973.
  24. ↑ Carrera P, Johnstone O, Nakamura A, Casanova J, Jäckle H, Lasko P (January 2000). "VASA mediates translation through interaction with a Drosophila yIF2 homolog". Molecular Cell. 5 (1): 181–7. doi:10.1016/s1097-2765(00)80414-1. hdl:11858/00-001M-0000-0012-F80E-6. PMID 10678180.
  25. ↑ Sengoku T, Nureki O, Nakamura A, Kobayashi S, Yokoyama S (April 2006). "Structural basis for RNA unwinding by the DEAD-box protein Drosophila Vasa". Cell. 125 (2): 287–300. doi:10.1016/j.cell.2006.01.054. PMID 16630817.
  26. ↑ Linder P, Lasko P (April 2006). "Bent out of shape: RNA unwinding by the DEAD-box helicase Vasa". Cell. 125 (2): 219–21. doi:10.1016/j.cell.2006.03.030. PMID 16630807.
  27. ↑ Lorsch JR (June 2002). "RNA chaperones exist and DEAD box proteins get a life". Cell. 109 (7): 797–800. doi:10.1016/s0092-8674(02)00804-8. PMID 12110176.
  28. ↑ Liu N, Han H, Lasko P (December 2009). "Vasa promotes Drosophila germline stem cell differentiation by activating mei-P26 translation by directly interacting with a (U)-rich motif in its 3' UTR". Genes & Development. 23 (23): 2742–52. doi:10.1101/gad.1820709. PMC 2788330. PMID 19952109.
  29. ↑ Styhler S, Nakamura A, Swan A, Suter B, Lasko P (May 1998). "vasa is required for GURKEN accumulation in the oocyte, and is involved in oocyte differentiation and germline cyst development". Development. 125 (9): 1569–78. doi:10.1242/dev.125.9.1569. PMID 9521895.
  30. ↑ Tanaka SS, Toyooka Y, Akasu R, Katoh-Fukui Y, Nakahara Y, Suzuki R, Yokoyama M, Noce T (April 2000). "The mouse homolog of Drosophila Vasa is required for the development of male germ cells". Genes & Development. 14 (7): 841–53. doi:10.1101/gad.14.7.841. PMC 316497. PMID 10766740.
  31. ↑ Mukai H, Watanabe H (March 1976). "Studies on the formation of germ cells in a compound ascidian Botryllus primigenus Oka". Journal of Morphology. 148 (3): 377–62. doi:10.1002/jmor.1051480306. PMID 943552. S2CID 46420722.
  32. ↑ Fabioux C, Corporeau C, Quillien V, Favrel P, Huvet A (May 2009). "In vivo RNA interference in oyster--vasa silencing inhibits germ cell development". The FEBS Journal. 276 (9): 2566–73. doi:10.1111/j.1742-4658.2009.06982.x. PMID 19476495.
  33. ↑ Miyake A, Saito T, Kashiwagi N, Ando D, Yamamoto A, Suzuki T, Nakatsuji N, Nakatsuji T (2006). "Cloning and pattern of expression of the shiro-uo vasa gene during embryogenesis and its roles in PGC development". The International Journal of Developmental Biology. 50 (7): 619–25. doi:10.1387/ijdb.062172am. PMID 16892175.
  34. ↑ Ikenishi K, Tanaka TS (October 1997). "Involvement of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) in the differentiation of primordial germ cells". Development, Growth & Differentiation. 39 (5): 625–33. doi:10.1046/j.1440-169x.1997.t01-4-00010.x. PMID 9338598. S2CID 20046434.
  35. ↑ Zhurov V, Terzin T, Grbić M (December 2004). "Early blastomere determines embryo proliferation and caste fate in a polyembryonic wasp". Nature. 432 (7018): 764–9. Bibcode:2004Natur.432..764Z. doi:10.1038/nature03171. PMID 15592416. S2CID 4341264.
  36. ↑ Hashimoto H, Sudo T, Mikami Y, Otani M, Takano M, Tsuda H, Itamochi H, Katabuchi H, Ito M, Nishimura R (November 2008). "Germ cell specific protein VASA is over-expressed in epithelial ovarian cancer and disrupts DNA damage-induced G2 checkpoint". Gynecologic Oncology. 111 (2): 312–9. doi:10.1016/j.ygyno.2008.08.014. PMID 18805576.
  37. ↑ Lavial F, Acloque H, Bachelard E, Nieto MA, Samarut J, Pain B (June 2009). "Ectopic expression of Cvh (Chicken Vasa homologue) mediates the reprogramming of chicken embryonic stem cells to a germ cell fate". Developmental Biology. 330 (1): 73–82. doi:10.1016/j.ydbio.2009.03.012. PMID 19324033.
  38. ↑ Bosch TC, David CN (May 1987). "Stem cells of Hydra magnipapillata can differentiate into somatic cells and germ line cells" (PDF). Developmental Biology. 121 (1): 182–191. doi:10.1016/0012-1606(87)90151-5.
  39. ↑ Shukalyuk AI, Golovnina KA, Baiborodin SI, Gunbin KV, Blinov AG, Isaeva VV (February 2007). "vasa-related genes and their expression in stem cells of colonial parasitic rhizocephalan barnacle Polyascus polygenea (Arthropoda: Crustacea: Cirripedia: Rhizocephala)". Cell Biology International. 31 (2): 97–108. doi:10.1016/j.cellbi.2006.09.012. PMID 17085060. S2CID 33510591.
  40. ↑ Fabioux C, Pouvreau S, Le Roux F, Huvet A (March 2004). "The oyster vasa-like gene: a specific marker of the germline in Crassostrea gigas". Biochemical and Biophysical Research Communications. 315 (4): 897–904. doi:10.1016/j.bbrc.2004.01.145. PMID 14985097.
  41. ↑ Swartz SZ, Chan XY, Lambert JD (February 2008). "Localization of Vasa mRNA during early cleavage of the snail Ilyanassa". Development Genes and Evolution. 218 (2): 107–13. doi:10.1007/s00427-008-0203-6. PMID 18214533. S2CID 9580109.
  42. ↑ Oyama A, Shimizu T (October 2007). "Transient occurrence of vasa-expressing cells in nongenital segments during embryonic development in the oligochaete annelid Tubifex tubifex". Development Genes and Evolution. 217 (10): 675–90. doi:10.1007/s00427-007-0180-1. hdl:2115/30348. PMID 17851685. S2CID 19507358.
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