Exogenous nucleic acids aggregate in non-P-body cytoplasmic granules when transfected into cultured cells
Received date: 08 Mar 2010
Accepted date: 31 Mar 2010
Published date: 01 Jun 2010
Copyright
To modulate gene expression in research studies or in potential clinical therapies, transfection of exogenous nucleic acids including plasmid DNA and small interference RNA (siRNA) are generally performed. However, the cellular processing and the fate of these nucleic acids remain elusive. By investigating the cellular behavior of transfected nucleic acids using confocal imaging, here we show that when siRNA was co-transfected into cultured cells with other nucleic acids, including single-stranded RNA oligonucleotides, single and double-stranded DNA oligonucleotides, as well as long double-stranded plasmid DNA, they all aggregate in the same cytoplasmic granules. Interestingly, the amount of siRNA aggregating in granules was found not to correlate with the gene silencing activity, suggesting that assembly of cytoplasmic granules triggered by siRNA transfection may be separable from the siRNA silencing event. Our results argue against the claim that the siRNA-aggregating granules are the functional site of RNA interference (RNAi). Taken together, our studies suggest that, independent of their types or forms, extraneously transfected nucleic acids are processed through a common cytoplasmic pathway and trigger the formation of a new type of cytoplasmic granules “ transfection granules” .
Huang HUANG , Na WEI , Yingfei XIONG , Feng YANG , Huaqiang FANG , Wenjun XIE , Zhuan ZHOU , Heping CHENG , Zicai LIANG , Quan DU . Exogenous nucleic acids aggregate in non-P-body cytoplasmic granules when transfected into cultured cells[J]. Frontiers in Biology, 2010 , 5(3) : 272 -281 . DOI: 10.1007/s11515-010-0047-0
1 |
Anderson P, Kedersha N (2006). RNA granules. J Cell Biol, 172(6): 803– 808
|
2 |
Anderson P, Kedersha N (2008). Stress granules: the Tao of RNA triage. Trends Biochem Sci, 33(3): 141– 150
|
3 |
Arimoto K, Fukuda H, Imajoh-Ohmi S, Saito H, Takekawa M (2008). Formation of stress granules inhibits apoptosis by suppressing stress-responsive MAPK pathways. Nat Cell Biol, 10(11): 1324– 1332
|
4 |
Berezhna S Y, Supekova L, Supek F, Schultz P G, Deniz A A (2006). siRNA in human cells selectively localizes to target RNA sites. Proc Natl Acad Sci U S A, 103(20): 7682– 7687
|
5 |
Bhattacharyya S N, Habermacher R, Martine U, Closs E I, Filipowicz W (2006). Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell, 125(6): 1111– 1124
|
6 |
Brengues M, Teixeira D, Parker R (2005). Movement of eukaryotic mRNAs between polysomes and cytoplasmic processing bodies. Science, 310(5747): 486– 489
|
7 |
Chiu Y L, Ali A, Chu C Y, Cao H, Rana T M (2004). Visualizing a correlation between siRNA localization, cellular uptake, and RNAi in living cells. Chem Biol, 11(8): 1165– 1175
|
8 |
Chu C Y, Rana T M (2006). Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54. PLoS Biol, 4(7): e210
|
9 |
Coller J, Parker R (2005). General translational repression by activators of mRNA decapping. Cell, 122(6): 875– 886
|
10 |
de Semir D, Petriz J, Avinyó A, Larriba S, Nunes V, Casals T, Estivill X, Aran J M (2002). Non-viral vector-mediated uptake, distribution, and stability of chimeraplasts in human airway epithelial cells. J Gene Med, 4(3): 308– 322
|
11 |
Eulalio A, Behm-Ansmant I, Schweizer D, Izaurralde E (2007). P-body formation is a consequence, not the cause, of RNA-mediated gene silencing. Mol Cell Biol, 27(11): 3970– 3981
|
12 |
Godbey W T, Wu K K, Mikos A G (1999). Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc Natl Acad Sci U S A, 96(9): 5177– 5181
|
13 |
Golzio M, Teissie J, Rols M P (2002). Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci U S A, 99(3): 1292– 1297
|
14 |
Jagannath A, Wood M J (2008) Localization of Double-stranded siRNA to Cytoplasmic P-Bodies Is Ago2-dependent and Results in Upregulation of GW182 and Ago2. Mol Biol Cell.
|
15 |
Jakymiw A, Lian S, Eystathioy T, Li S, Satoh M, Hamel J C, Fritzler M J, Chan E K (2005). Disruption of GW bodies impairs mammalian RNA interference. Nat Cell Biol, 7(12): 1267– 1274
|
16 |
Jakymiw A, Pauley K M, Li S, Ikeda K, Lian S, Eystathioy T, Satoh M, Fritzler M J, Chan E K (2007). The role of GW/P-bodies in RNA processing and silencing. J Cell Sci, 120(Pt 8): 1317– 1323
|
17 |
Kedersha N, Anderson P (2007). Mammalian stress granules and processing bodies. Methods Enzymol, 431: 61– 81
|
18 |
Lian S, Fritzler M J, Katz J, Hamazaki T, Terada N, Satoh M, Chan E K (2007). Small interfering RNA-mediated silencing induces target-dependent assembly of GW/P bodies. Mol Biol Cell, 18(9): 3375– 3387
|
19 |
Liu J, Rivas F V, Wohlschlegel J, Yates J R 3rd, Parker R, Hannon G J (2005a). A role for the P-body component GW182 in microRNA function. Nat Cell Biol, 7(12): 1261– 1266
|
20 |
Liu J, Valencia-Sanchez M A, Hannon G J, Parker R (2005b). MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nat Cell Biol, 7(7): 719– 723
|
21 |
Lukacs G L, Haggie P, Seksek O, Lechardeur D, Freedman N, Verkman A S (2000). Size-dependent DNA mobility in cytoplasm and nucleus. J Biol Chem, 275(3): 1625– 1629
|
22 |
Marcusson E G, Bhat B, Manoharan M, Bennett C F, Dean N M (1998). Phosphorothioate oligodeoxyribonucleotides dissociate from cationic lipids before entering the nucleus. Nucleic Acids Res, 26(8): 2016– 2023
|
23 |
Niidome T, Huang L (2002). Gene therapy progress and prospects: nonviral vectors. Gene Ther, 9(24): 1647– 1652
|
24 |
Pauley K M, Eystathioy T, Jakymiw A, Hamel J C, Fritzler M J, Chan E K (2006). Formation of GW bodies is a consequence of microRNA genesis. EMBO Rep, 7(9): 904– 910
|
25 |
Sen G L, Blau H M (2005). Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nat Cell Biol, 7(6): 633– 636
|
26 |
Serman A, Le Roy F, Aigueperse C, Kress M, Dautry F, Weil D (2007). GW body disassembly triggered by siRNAs independently of their silencing activity. Nucleic Acids Res, 35(14): 4715– 4727
|
27 |
Shimizu N, Kamezaki F, Shigematsu S (2005). Tracking of microinjected DNA in live cells reveals the intracellular behavior and elimination of extrachromosomal genetic material. Nucleic Acids Res, 33(19): 6296– 6307
|
28 |
St Johnston D (2005). Moving messages: the intracellular localization of mRNAs. Nat Rev Mol Cell Biol, 6(5): 363– 375
|
29 |
Vickers T A, Lima W F, Wu H, Nichols J G, Linsley P S, Crooke S T (2009). Off-target and a portion of target-specific siRNA mediated mRNA degradation is Ago2 ‘ Slicer’ independent and can be mediated by Ago1. Nucleic Acids Res, 37(20): 6927– 6941
|
30 |
Wells D J (2004). Gene therapy progress and prospects: electroporation and other physical methods. Gene Ther, 11(18): 1363– 1369
|
/
〈 | 〉 |