Biopolym. Cell. 2011; 27(6):459-464 .
Structure and Function of Biopolymers
Identification of novel TDRD7 isoforms
1Skorokhod O. M., 1Gudkova D. O., 1Filonenko V. V.
  1. State Key Laboratory of Molecular and Cellular Biology
    Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680

Abstract

Aim. The aim of our study was to investigate the tudor domain-containing protein 7 (TDRD7 ) subcellular localization, which could be linked to diverse functions of this protein within the cell. Methods. In this study we employed cell imaging technique for detecting TDRD7 subcellular localization, Western blot analysis of HEK293 cell fractions with anti-TDRD7 monoclonal antibodies and bioinformatical search of possible TDRD7 isoforms in Uniprot, Ensemble, UCSC databases. Results. We have observed specific TDRD7-containing structures in cytoplasm as well as in the nucleus in HEK293 cells. The Western blot analysis of subcellular fractions (cytoplasm, mitochondria, nucleus) allowed us to detect three lower immunoreactive bands, with the aproximate molecular weight of 130, 110 and 60 kDa (we termed them as TDRD7β, TDRD7γ and TDRD7δ) and specific subcellular localization. The bioinformatical analysis of TDRD7 primary structure allowed us to determine two alternative transcripts from TDRD7 gene coding for proteins with calculated molecular weight of 130 and 60 kDa. Conclusion. The presented data demonstrate the existence at protein level of potential TDRD7 isoforms: TDRD7β, TDRD7 γ and TDRD7δ. The expression profile of these splice variants and their role in cells remains to be elucidated.
Keywords: TDRD7, S6 kinase, isoform, piRNAs, translation

References

[1] Fingar D. C., Salama S., Tsou C., Harlow E., Blenis J. Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E Genes Dev 2002 16, N 12 P.1472–1487.
[2] Inoki K., Ouyang H., Li Y., Guan K.-L. Signaling by target of rapamycin proteins in cell growth control Microbiol. Mol. Biol. Rev 2005 69, N 1 P. 79–100.
[3] Panasyuk G., Nemazanyy I., Filonenko V., Zhyvoloup A. Largescale yeast transformation in low-percentage agarose medium BioTechniques 2004 36, N 1 P. 40–44.
[4] Hirose T., Kawabuchi M., Tamaru T., Okumura N., Nagai K., Okada M. Identification of tudor repeat associator with PCTAIRE 2 (Trap). A novel protein that interacts with the N-terminal domain of PCTAIRE 2 in rat brain Eur. J. Biochem 2000 267, N 7 P. 2113–2121.
[5] Chuma S., Hosokawa M., Kitamura K., Kasai S., Fujioka M., Hiyoshi M., Takamune K., Noce T., Nakatsuji N. 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. USA 2006 103, N 43 P. 15894–15899.
[6] Vasileva A., Tiedau D., Firooznia A., Muller-Reichert T., Jessberger R. Tdrd6 is required for spermiogenesis, chromatoid body architecture, and regulation of miRNA expression Curr. Biol 2009 19, N 8 P. 630–639.
[7] Lachke S. A., Alkuraya F. S., Kneeland S. C., Ohn T., Aboukhalil A., Howell G. R., Saadi I., Cavallesco R., Yue Y., Tsai A. C., Nair K. S., Cosma M. I., Smith R. S., Hodges E., Alfadhli S. M., Al-Hajeri A., Shamseldin H. E., Behbehani A., Hannon G. J., Bulyk M. L., Drack A. V., Anderson P. J., John S. W., Maas R. L. Mutations in the RNA granule component TDRD7 cause cataract and glaucoma Science 2011 331, N 6024 P. 1571– 1576.
[8] Hosokawa M., Shoji M., Kitamura K., Tanaka T., Noce T., Chuma S., Nakatsuji N. Tudor-related proteins TDRD1/MTR-1, TDRD6 and TDRD7/TRAP: domain composition, intracellular localization, and function in male germ cells in mice Dev. Biol 2007 301, N 1 P. 38–52.
[9] Zukerberg R., Patrick N., Nikolic M., Humbert S., Wu C., Lanier L., Gertler F., Vidal M., Van Etten R., Tsai L. Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth Neuron 2000 26, N 3 P. 633–646.
[10] Kotaja N., Bhattacharyya S. N., Jaskiewicz L., Kimmins S., Parvinen M., Filipowicz W., Sassone-Corsi P. The chromatoid body of male germ cells: similarity with processing bodies and presence of Dicer and microRNA pathway components Proc. Natl Acad. Sci. USA 2006 103, N 8 P. 2647–2652.
[11] Yamochi T., Nishimoto I., Tsukasa Okuda T., Matsuoka M. ik31/Cables is associated with Trap and Pctaire2 Biochem. Biophys. Res. Commun 2001 286, N 5 P. 1045–1050.
[12] Conte N., Delaval B., Ginestier C., Ferrand A., Isnardon D., Larroque C., Prigent C., Seraphin B., Jacquemier J., Birnbaum D. TACC1-chTOG-Aurora A protein complex in breast cancer Oncogene 2003 22, N 50 P. 8102–8116.
[13] Richter J. Think globally, translate locally: What mitotic spindles and neuronal synapses have in common Proc. Natl Acad. Sci. USA 2001 98, N 13 P. 7069–7071.
[14] Selenko P., Sprangers R., Stier G., Buhler D., Fischer U., Sattler M. SMN tudor domain structure and its interaction with the Sm proteins Nat. Struct. Biol 2001 8, N 1 P. 27–31.
[15] Ponting C. P. Tudor domains in proteins that interact with RNA Trends Biochem. Sci 1997 22, N 2 P. 51–52.
[16] Amikura R., Hanyu K., Kashikawa M., Kobayashi S. Tudor protein is essential for the localization of mitochondrial RNAs in polar granules of Drosophila embryos Mech. Dev 2001 107, N 1–2 P. 97–104.
[17] 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., Halazonetis T. D. Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks Nature 2004 432, N 7015 P. 406–411.
[18] Jin J., Xie X., Chen C., Park J. G., Stark C., James D. A., Olhovsky M., Linding R., Mao Y., Pawson T. Eukaryotic protein domains as functional units of cellular evolution Sci. Signal 2009 2, N 98 P. 1–19.
[19] Cote J., Richard S. Tudor domains bind symmetrical dimethylated arginines J. Biol. Chem 2005 280, N 31 P. 28476– 28483.
[20] Charier G., Couprie J., Alpha-Bazin B., Meyer V., Quemeneur E., Guerois R., Callebaut I., Gilquin B., Zinn-Justin S. The Tudor tandem of 53BP1: a new structural motif involved in DNA and RG-rich peptide binding Structure 2004 12, N 9 P. 1551–1562.
[21] Thomson T., Lasko P. Tudor and its domains: germ cell formation from a Tudor perspective Cell Res 2005 15, N 4 P. 281–291.
[22] Callebaut I., Mornon J.-P. LOTUS, a new domain associated with small RNA pathways in the germline Bioinformatics 2010 26, N 9 P. 1140–1144.
[23] Lee C., Atanelov L., Modrek B., Xing Y. ASAP: the alternative splicing annotation project Nucleic Acids Res 2003 31, N 1 P. 101–105.
[24] Skorokhod O., Nemazanyy I., Breus O., Filonenko V., Panasyuk G. Generation and characterization of monoclonal antibodies to TDRD7 protein Hybridoma 2008 27, N 3 P. 211–216.
[25] Gudkova D. O., Panasyuk G. G., Nemazanyy I. O., Filonenko V. V. Novel antibodies against RCD-8 as a tool to study processing bodies Biopolym. Cell 2010 26, N 6 P. 512–516.
[26] Zhyvoloup A., Nemazanyy I., Babich A., Panasyuk G., Pobigailo N., Vudmaska M., Naidenov V., Kukharenko O., Palchevskii S., Savinska L., Ovcharenko G., Verdier F., Valovka T., Fenton T., Rebholz H., Wang M. L., Shepherd P., Matsuka G., Filonenko V., Gout I. T. Molecular cloning of CoA Synthase. The missing link in CoA biosynthesis J. Biol. Chem 2002 277, N 25 P. 22107– 22110.
[27] Wieckowski M. R., Giorgi C., Lebiedzinska M., Duszynski J., Pinton P. Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells Nat. Protoc 2009 4, N 11 P. 1582–1590.