Analysis of C. elegans VIG-1 Expression

  • Shin, Kyoung-Hwa (School of Life Sciences, Chungbuk National University) ;
  • Choi, Boram (School of Life Sciences, Chungbuk National University) ;
  • Park, Yang-Seo (School of Life Sciences, Chungbuk National University) ;
  • Cho, Nam Jeong (School of Life Sciences, Chungbuk National University)
  • Received : 2008.06.02
  • Accepted : 2008.09.09
  • Published : 2008.12.31

Abstract

Double-stranded RNA (dsRNA) induces gene silencing in a sequence-specific manner by a process known as RNA interference (RNAi). The RNA-induced silencing complex (RISC) is a multi-subunit ribonucleoprotein complex that plays a key role in RNAi. VIG (Vasa intronic gene) has been identified as a component of Drosophila RISC; however, the role VIG plays in regulating RNAi is poorly understood. Here, we examined the spatial and temporal expression patterns of VIG-1, the C. elegans ortholog of Drosophila VIG, using a vig-1::gfp fusion construct. This construct contains the 908-bp region immediately upstream of vig-1 gene translation initiation site. Analysis by confocal microscopy demonstrated GFP-VIG-1 expression in a number of tissues including the pharynx, body wall muscle, hypodermis, intestine, reproductive system, and nervous system at the larval and adult stages. Furthermore, western blot analysis showed that VIG-1 is present in each developmental stage examined. To investigate regulatory sequences for vig-1 gene expression, we generated constructs containing deletions in the upstream region. It was determined that the GFP expression pattern of a deletion construct (${\Delta}-908$ to -597) was generally similar to that of the non-deletion construct. In contrast, removal of a larger segment (${\Delta}-908$ to -191) resulted in the loss of GFP expression in most cell types. Collectively, these results indicate that the 406-bp upstream region (-596 to -191) contains essential regulatory sequences required for VIG-1 expression.

Keywords

C. elegans;RNA-induced silencing complex;RNA interference;VIG-1

Acknowledgement

Supported by : Korea Research Foundation

References

  1. Caudy, AA, Myers, M., Hannon, G.J., and Hammond, S.M. (2002). Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev. 16,2491-2496 https://doi.org/10.1101/gad.1025202
  2. Kim, Y.H., Song, H.-O., Ko, K.M., Singaravelu, G., Jee C., Kang, J., and Ahnn, J. (2008). A novel calcineurin-interacting protein, CNP-3, modulates calcineurin deficient phenotypes in Caenorhabditis elegans. Mol. Cells 25, 566-571
  3. Meister, G., and Tuschl, 1. (2004). Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343-349 https://doi.org/10.1038/nature02873
  4. Bernstein, E., Gaudy, A.A., Hammond, S.M., and Hannon, G.J. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363-366 https://doi.org/10.1038/35053110
  5. Tijsterman, M., Kelting, R.F., and Plasterk, RHA (2002). The genetics of RNA silencing. Annu. Rev. Genet. 36, 489-519 https://doi.org/10.1146/annurev.genet.36.043002.091619
  6. Caudy, AA, Kelting, R.F., Hammond, S.M., Denli, AM., Bathoorn, AMP., Tops, B.B.J., Silva, J.M., Myers, M.M., Hannon, G.J., and Plasterk, RHA (2003). A micrococcal nuclease homologue in RNAi effector complexes. Nature 425, 411-414 https://doi.org/10.1038/nature01956
  7. Fire, A, Xu, S., Montgomery, MK, Kostas, SA, Driver, S.E., and Mello, C.C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811 https://doi.org/10.1038/35888
  8. Kennerdell, J.R., and Carthew, RW. (1998). Use of dsRNAmediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95, 1017-1026 https://doi.org/10.1016/S0092-8674(00)81725-0
  9. Mello, C.C., Cramer, J.M., Stinchcomb, D., and Ambros, V. (1991). Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959-3970
  10. Tang, G. (2005). siRNA and miRNA: an insight into RISCs. Trends Biochem. Sci. 30, 106-114 https://doi.org/10.1016/j.tibs.2004.12.007
  11. Timmons, L., and Fire, A (1998). Specific interference by ingested dsRNA Nature 395, 854 https://doi.org/10.1038/27579
  12. Hannon, G.J. (2002). RNA interference. Nature 418, 244-251 https://doi.org/10.1038/418244a
  13. Knight, SW., and Bass, B.L. (2001). A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans. Science 293, 2269-2271 https://doi.org/10.1126/science.1062039
  14. Dudley, N.R., Labbe, J.-C., and Goldstein B. (2002). Using RNA interference to identify genes required for RNA interference. Proc. Natl. Acad. Sci. USA 99, 4191-4196
  15. He, L., and Hannon, G.J. (2004). MicroRNAs: small RNAs with a big role in gene regulation. Nat. Rev. Genet. 5,522-531 https://doi.org/10.1038/nrg1379
  16. MacRae, I.J., Zhou, K., Li, F., Repic, A, Brooks, AN., Cande, W.Z., Adams, p.o., and Doudna, JA (2006). Structural basis for double-stranded RNA processing by Dicer. Science 311,195-198 https://doi.org/10.1126/science.1121638
  17. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94
  18. Sontheimer, E.J. (2005). Assembly and function of RNA silencing complexes. Nat. Rev. Mol. Cell. BioI. 6, 127-138 https://doi.org/10.1038/nrm1568
  19. Wianny, F., and Zernicka-Goetz, M. (2000). Specific interference with gene function by double-stranded RNA in early mouse development. Nat. Cell BioI. 2, 70-75 https://doi.org/10.1038/35000016