Functional Analysis of Nuage Component Maelstrom. Tan L.Z 1 and Kai T 2. Singapore

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1 Functional Analysis of Nuage Component Maelstrom Tan L.Z 1 and Kai T 2. Department of Biological Sciences, Faculty of Science, National University of Singapore Abstract This paper aims to address the functional mechanisms of germline-specific nuage component, Maelstrom. Malestrom (mael) has been shown to shuttle between cytoplasm and nucleus, suggesting its implications in its involvement in DNA silencing via pirna and mirna pathways in both cytoplasmic and nucleus region. The exact mechanism of mael shuttling into nucleus remains unknown; however, using Drosophila melangaster as a model system, and employment of molecular techniques such as immunostaining, DNA transformation, cell transfection, and western blotting, a proposed model where mael entry into nucleus is regulated by phosphorylation mechanism via p38 MAPK signaling pathway is illustrated in this paper. Heat sensitive p38 protein kinase level has been shown to increase in heat shock treated wild type Drosophila. Colocalisation of p38 and mael at perinuclear region has also been observed, suggesting phosphorylation of mael by p38 could potentially take place in perinuclear region. Results using transgenic flies with altered mael gene sequence further illustrates phosphorylation as a potential way of regulating mael shuttling into nucleus and a possible phosphorylation site in mael gene sequence is also being explored here. Results 1 Student 2 Assistant Professor

2 obtained have not disproved the proposed model and follow-up investigative work is required. Brief outline of what has been done in the course of the entire research Increase in heat sensitive p38 protein kinase level in yw Drosophila after one hour heat shock as compared to yw flies without heat shock was observed using western blot method. Antibody staining of flies that had undergone heat stress treatment showed colocalisation of p38 and maelstrom in the perinuclear region, suggesting perinuclear region might be the site where mael is phosphorylated by p38 mitogenactivated protein kinase (MAPK) when subjected to heat stress. p38 Fig 1. Lighter protein band observed in yw flies without heat shock treatment (-HS) compared to band observed in yw with heat shock treatment (+HS), suggesting higher level of p38 in ovaries of heat shock treated yw. Actin Fig 2. Antibody staining with anti-p38 (green) and anti-mael (red) reveals colocalisation of mael and p38 in perinuclear region, and also shuttling of some mael into the nucleus of germ cells.

3 Furthermore, western blotting done on transgenic flies revealed that slower mobility band in evident transgenic flies with mael-fl (full length). A possible theory for the observed difference in mobility bands could be due to differences in phosphorylation state. If phosphorylation is indeed the key in mael shuttling into nucleus, S138 of mael sequence could potentially be the site of phosphorylation Based on results obtained, it is hypothesized that phosphorylation process might potentially be the regulating mechanism for mael entry into the nucleus. Possible functions of mael in the germ cell nucleus could be related to its involvement in mediating piwi proteins in nucleus to form piwi-pirna complexes for silencing of mobile elements. Reference Aravin, A.A., Lagos-Quintana, M., Yalcin, A., Zavolan, M., Marks, D., Snyder, B., Gaasterland, T., Meyer, J., Tuschl, T The Small RNA Profile during Drosophila melanogaster Development. Dev. Cell 5: Brennecke, J., Aravin, A.A., Stark, A., Dus, M., Kellis, M., Sachidanandam, R. and Hannon, G.J. (2007). Discrete small RNA-generating loci as master regulators of transposon acitivity in Drosophila. Cell 128: Findley, S.D., Tamanaha, M., Clegg N.J. and Ruohola-Baker (2003). Maelstrom, a Drosophila spindle-class gene, encodes a protein that colocalizes with Vasa and RDE1/AGO1 homologu, Aubergine, in nuage. Development 130: Klattenhoff, C. and Theurkauf, W. (2008). Biogenesis and germline functions of pirnas. Development 135: 3-9.

4 Kotaja, N. and Sassone-Corsi, P. (2007). The chromotoid body: a germ-cell-specific RNA- processing center. Nature Reviews Molecular Cell Biology 8: Han SJ et al, (1998). Molecular cloning and characterization of a Drosophila p38 mitogen-activated protein kinase. J Bio Chem 273: Lim, A.K. and Kai, T. (2007). Unique germ-line organelle, nuage, functions to repress selfish genetic elements in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 104: Mahowald, AP, (1971). Polar granules of Drosophila. III. The continuity of polar granules during the life cycle of Drosophila. J Exp Zool, 176: O Donelle and Boeke, (2007). Mighty piwis defend the germline against genomic intruders. Cell 129: Pek, Lim and Kai, (2009). Maelstrom differentially regulates micrornas and pirnas to control germline proliferation and differentiation in Drosophila Soper SF, van der Heijden GW, Hardiman TC, Goodheart M, Martin SL, de Boer P, Bortvin A, (2008) Mouse maelstrom, a component of nuage, is essential for spermatogenesis and transposon repression in meiosis. Dev Cell Aug;15(2):

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