Animal cells and systems

The Korean Society for Integrative Biology (한국통합생물학회)
권호 표지

Expression of PACT and EIF2C2, Implicated in RNAi and MicroRNA Pathways, in Various Human Cell Lines

  • Lee, Yong-Sun (Department of Biochemistry and Molecular Genetics, University of Virginia, Institute of Molecular Biology and Genetics, School of Biological Sciences, Seoul National University) ;
  • Jeon, Yesu (Department of Biochemistry and Molecular Genetics, University of Virginia, Institute of Molecular Biology and Genetics, School of Biological Sciences, Seoul National University) ;
  • Park, Jong-Hoon (Department of Biochemistry and Molecular Genetics, University of Virginia, Institute of Molecular Biology and Genetics, School of Biological Sciences, Seoul National University) ;
  • Hwang, Deog-Su (Institute of Molecular Biology and Genetics, School of Biological Sciences, Seoul National University) ;
  • Dutta, Anindya (Department of Biochemistry and Molecular Genetics, University of Virginia)
  • Published : 2004.09.01
Download PDF
⟨ Previous Next ⟩

Abstract

MicroRNA and siRNA (small interfering RNA), representative members of small RNA, exert their effects on target gene expression through association with protein complexes called miRNP (microRNA associated ribonucleoproteins) and RISC (RNA induced silencing complex), respectively. Although the protein complexes are yet to be fully characterized, human EIF2C2 protein has been identified as a component of both miRNP and RISC. In this report, we raised antiserum against EIF2C2 in order to begin understanding the protein complexes. An immunoblot result indicates that EIF2C2 protein is ubiquitously expressed in a variety of cell lines from human and mouse. EIF2C2 protein exists in both cellular compartments, as indicated by an immunoblot assay with a nuclear extract and a cytosolic fraction (S100 fraction) from HeLa S3 lysate. Depletion of EIF2C1 or EIF2C2 protein resulted in a decrease of microRNA, suggesting a possible role of these proteins in microRNA stability or biogenesis. We also prepared antiserum against dsRNA binding protein PACT, whose homologs in C. elegans and Drosophila are known to have a role in the RNAi (RNA interference) pathway. The expression of PACT protein was also observed in a wide range of cell lines.

Keywords

References

  1. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: 281-297 https://doi.org/10.1016/S0092-8674(04)00045-5
  2. Billy E, Brondani V, Zhang H, Muller U and Filipowicz W (2001) Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc Natl Acad Sci USA 98: 14428-14433 https://doi.org/10.1073/pnas.261562698
  3. Carmell MA, Xuan Z, Zhang MQ and Hannon GJ (2002) The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev 16: 2733-2742 https://doi.org/10.1101/gad.1026102
  4. Cerutti L, Mian N, and Bateman A (2000) Domains in gene silencing and cell differentiation proteins: the novel PAZ domain and redefinition of the Piwi domain. Trends Biochem Sci 25: 481-482 https://doi.org/10.1016/S0968-0004(00)01641-8
  5. Dignam JD, Lebovitz RM, and Roeder RG (1983b) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acid Res 11: 1475-1489 https://doi.org/10.1093/nar/11.5.1475
  6. Doench JG, Petersen CP, and Sharp PA (2003) siRNAs can function as miRNAs. Genes Dev 17: 438-442 https://doi.org/10.1101/gad.1064703
  7. Doi N, Zenno S, Ueda R, Ohki-Hamazaki H, Ui-Tei K, and Saigo K (2003) Shortinterfering-RNA-mediated gene silencing in mammalian cells requires Dicer and elF2C translation initiation factors. Curr Biol 13: 41-46 https://doi.org/10.1016/S0960-9822(02)01394-5
  8. Dykxhoorn DM, Novina CD, and Sharp PA (2003) Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 4: 457-467 https://doi.org/10.1038/nrm1129
  9. Ekwall K (2004) The RITS complex-A direct link between small RNA and heterochromatin. Mol Cell 13: 304-305 https://doi.org/10.1016/S1097-2765(04)00057-7
  10. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, and Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494-498 https://doi.org/10.1038/35078107
  11. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, and Mello CC (1998) Potent and specific genetic interference by double-stmnded RNA in Caenorhabditis elegans. Nature 391: 806-811 https://doi.org/10.1038/35888
  12. Fukagawa T, Nogami M, Yoshikawa M, Ikeno M, Okazaki T, Takami Y, Nakayama T, and Oshimura M (2004) Dicer is essential for formation of the heterochromatin structure in vertebrate cells. Nat Cell Biol 6: 784-791 https://doi.org/10.1038/ncb1155
  13. Gupta V and Patel RC (2002) Proapoptotic protein PACT is expressed at high levels in colonic epithelial cells in mice. Am J Physiol Gastrointest Liver Physiol 283: G801-808 https://doi.org/10.1152/ajpgi.00498.2001
  14. Hammond SM, Bernstein E, Beach D, and Hannon GJ (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404: 293-296 https://doi.org/10.1038/35005107
  15. Hammond SM, Boettcher S, Caudy AA, Kobayashi R, and Hannon GJ (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293: 1146-1150 https://doi.org/10.1126/science.1064023
  16. Hannon GJ (2002) RNA interference. Nature 418: 244-251 https://doi.org/10.1038/418244a
  17. Hutvagner G, McLachlan J, Pasquinelli AE, Balint E, Tuschl T, and Zamore PD (2001) A cellular function for the RNAinterference enzyme dicer in the maturation of the let-7 small temporal RNA. Science 293: 834-838 https://doi.org/10.1126/science.1062961
  18. Hutvagner G and Zamore PD (2002) A microRNA in a multipleturnover RNAi enzyme complex. Science 297: 2056-2060 https://doi.org/10.1126/science.1073827
  19. Ishizuka A, Siomi MC, and Siomi H (2002) A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev 16: 2497-2508 https://doi.org/10.1101/gad.1022002
  20. Lee RC, Feinbaum RL, and Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75: 843-854 https://doi.org/10.1016/0092-8674(93)90529-Y
  21. Lingel A, Simon B, Izaurralde E, and Sattler M (2003) Structure and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain. Nature 426: 465-469 https://doi.org/10.1038/nature02123
  22. Liu Q, Rand TA, Kalidas S, Du F, Kim HE, Smith DP, and Wang X (2003) R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science 301: 1921-1925 https://doi.org/10.1126/science.1088710
  23. Martinez J, Patkaniowska A, Urlaub H, Luhrmann Rand Tuschl T (2002) Singlestranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110: 563-574 https://doi.org/10.1016/S0092-8674(02)00908-X
  24. Mourelatos Z, Dostie J, Paushkin S, Sharma A, Charroux B, Abel L, Rappsilber J, Mann M, and Dreyfuss G (2002) miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev 16: 720-728 https://doi.org/10.1101/gad.974702
  25. Okamura K, Ishizuka A, Siomi H, and Siomi MC (2004) Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev 18: 1655-1666 https://doi.org/10.1101/gad.1210204
  26. Patel RC and Sen GC (1998) PACT, a protein activator of the interferon-induced protein kinase, PKR. EMBO J 17: 4379-4390 https://doi.org/10.1093/emboj/17.15.4379
  27. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, and Ruvkun G (2000) The 21nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403: 901-906 https://doi.org/10.1038/35002607
  28. Sambrook J, Fritsch EF, and Maniatis T (1989) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor
  29. Sasaki T, Shiohama A, Minoshima S, and Shimizu N (2003) Identification of eight members of the Argonaute family in the human genome small star, filled. Genomics 82: 323-330 https://doi.org/10.1016/S0888-7543(03)00129-0
  30. Saxena S, Jonsson ZO, and Dutta A (2003) Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells. J Biol Chem 278: 44312-44319 https://doi.org/10.1074/jbc.M307089200
  31. Schwarz DS and Zamore PD (2002) Why do miRNAs live in the miRNP? Genes Dev 16: 1025-1031 https://doi.org/10.1101/gad.992502
  32. Song JJ, Liu J, Tolia NH, Schneiderman J, Smith SK, Martienssen RA, Hannon GJ, and Joshua-Tor L (2003) The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes. Nat Struct Biol 10: 1026-1032 https://doi.org/10.1038/nsb1016
  33. Stark GR, Kerr IM, Williams BR, Silverman RH, and Schreiber RD (1998) How cells respond to interferons. Annu Rev Biochem 67: 227-264 https://doi.org/10.1146/annurev.biochem.67.1.227
  34. Tabara H, Sarkissian M, Kelly WG, Fleenor J, Grishok A, Timmons L, Fire A, and Mello CC (1999) The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99: 123-132 https://doi.org/10.1016/S0092-8674(00)81644-X
  35. Tabara H, Yigit E, Siomi H, and Mello CC (2002) The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans. Cell 109: 861-871 https://doi.org/10.1016/S0092-8674(02)00793-6
  36. Tang G, Reinhart BJ, Bartel DP, and Zamore PD (2003) A biochemical framework for RNA silencing in plants. Genes Dev 17: 49-63 https://doi.org/10.1101/gad.1048103
  37. Yang S, Tutton S, Pierce E, and Yoon K (2001) Specific doublestranded RNA interference in undifferentiated mouse embryonic stem cells. Mol Cell Biol 21: 7807-7816 https://doi.org/10.1128/MCB.21.22.7807-7816.2001
  38. Zeng Y and Cullen BR (2002) RNA interference in human cells is restricted to the cytoplasm. RNA 8: 855-860 https://doi.org/10.1017/S1355838202020071
  39. Zeng Y, Wagner EJ, and Cullen BR (2002) Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell 9: 1327-1333 https://doi.org/10.1016/S1097-2765(02)00541-5