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Frontiers in Biology

ISSN 1674-7984

ISSN 1674-7992(Online)

CN 11-5892/Q

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Front Biol    2010, Vol. 5 Issue (3) : 272-281    https://doi.org/10.1007/s11515-010-0047-0
RESEARCH ARTICLE
Exogenous nucleic acids aggregate in non-P-body cytoplasmic granules when transfected into cultured cells
Huang HUANG1, Na WEI1, Yingfei XIONG1,2, Feng YANG1, Huaqiang FANG1, Wenjun XIE1, Zhuan ZHOU1, Heping CHENG1, Zicai LIANG1(), Quan DU1()
1. Institute of Molecular Medicine, Peking University, Beijing 100871, China; 2. Institute of Neurosciences, The Fourth Military Medical University, Xi'an 710032, China
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Abstract

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” .
Keywords small interference RNA (siRNA)      nucleic acids      P-body      RNA interference (RNAi)      transfection     
Corresponding Author(s): LIANG Zicai,Email:liangz@pku.edu.cn; DU Quan,Email:quan.du@pku.edu.cn   
Issue Date: 01 June 2010
 Cite this article:   
Huang HUANG,Na WEI,Yingfei XIONG, et al. Exogenous nucleic acids aggregate in non-P-body cytoplasmic granules when transfected into cultured cells[J]. Front Biol, 2010, 5(3): 272-281.
 URL:  
https://academic.hep.com.cn/fib/EN/10.1007/s11515-010-0047-0
https://academic.hep.com.cn/fib/EN/Y2010/V5/I3/272
oligonucleotidessequence
siRNA-15￠ -UUCUCCGAACGUGUCACGUtt
(sense 3￠ -Alexa-647)ttAAGAGGCUUGCACAGUGCA-5￠
siRNA-25￠ -GCGACUCCAGAAGUUGUAAtt
(sense 5￠ -FAM)ttCGCUGAGGUCUUCAACAUU-5￠
siRNA-35￠ -GCGGAUCUGUGUUGCUCAUtt
(sense 5￠ -FAM)ttCGCCUAGACACAACGAGUA-5￠
siRNA-45￠ -CCCUGCAGUACAACUCCAUtt
ttGGGACGUCAUGUUGAGGUA-5￠
ssRNA-1 (5￠ -FAM)5￠ -UCGGGAAAUUUCUCUAUUAtt
ssRNA-2 (5￠ -FAM)5￠ -GCAGCACGACUUCUUCAAGtt
dsDNA-15￠ -GGAGTGTAACGATTACATCTT
(upper 5￠ -FAM)TTCCTCACATTGCTAATGTAG-5￠
dsDNA-25￠ -GTAATGGATGATTATGGAATT
(upper 5￠ -FAM)TTCATTACCTACTAATACCTT-5￠
ssDNA-1 (5￠ -FAM)5￠ -GGAGTGTAACGATTACATCTT
ssRNA-2 (5￠ -FAM)5￠ -GTAATGGATGATTATGGAATT
Ago1 upstream primer5￠ -CTGGCAAGAATTCTATATGGGATGGAAGCGGGAC
Ago1 downstream primer5￠ -TGCCTCACCGCGGTCCAGTGAGGTAACAGCGTTCTG
Ago2 upstream primer5￠ -CTGGCAAGAATTCGCGCCACCATGTACTCGGGAGC
Ago2 downstream primer5￠ -TGCCTCACCGCGGGAATCCCACTCGGTACACAATCG
DCP1 upstream primer5￠ -CTGGCAAGAATTCGATTCAAGATGGAGGCGCTGAGT
DCP1 downstream primer5￠ -TGCCTCACCGCGGTGGGGCTCTGCCTTTAGACTTA
Tab.1  Oligonucleotides in the study
Fig.1  Transfected siRNA aggregates in cytoplasmic granules which are not colocalized with RISC or P body components. Fluorescently labeled siRNA (Alexa-647-siRNA-1) was co-transfected into cultured HEK293 cells with a vector expressing the GFP-fused DCP1, Ago-1 or Ago-2 gene. An original GFP expressing plasmid was included as control. A: Distributions of siRNA and fusion genes in living cells 24 h after the transfections, using laser confocal microscopy. B: siRNA was transfected at final concentration of 13 nmol/L and fusion reporter vectors were transfected at final concentration of 0.33 ng/μ L. All of the transfections were performed using Lipofectamine 2000. The area covered by random overlapping of the two types of fluorescence was set as control, and the area covered by co-labeling was compared to control to quantitatively analyze the significance of co-aggregation.
Fig.1  Transfected siRNA aggregates in cytoplasmic granules which are not colocalized with RISC or P body components. Fluorescently labeled siRNA (Alexa-647-siRNA-1) was co-transfected into cultured HEK293 cells with a vector expressing the GFP-fused DCP1, Ago-1 or Ago-2 gene. An original GFP expressing plasmid was included as control. A: Distributions of siRNA and fusion genes in living cells 24 h after the transfections, using laser confocal microscopy. B: siRNA was transfected at final concentration of 13 nmol/L and fusion reporter vectors were transfected at final concentration of 0.33 ng/μ L. All of the transfections were performed using Lipofectamine 2000. The area covered by random overlapping of the two types of fluorescence was set as control, and the area covered by co-labeling was compared to control to quantitatively analyze the significance of co-aggregation.
Fig.2  No correlation was found between granule-aggregated siRNA and gene silencing efficacy. A fluorescently labeled siRNA (FAM-siRNA-2) and an effective non-labeled siRNA (siRNA-4) targeting a luciferase fusion gene were individually diluted and transfected into HEK293 cells, together with the target reporter vector. To keep the siRNA concentration constant, an irrelevant siRNA was added into the dilutions. A: Distributions of the labeled siRNA 4 h after the transfections. B: Granule-aggregated siRNA was quantified by measuring the fluorescence intensity of the siRNA-aggregating granules, and was compared with the gene silencing efficacy of the unlabeled siRNA.
Fig.2  No correlation was found between granule-aggregated siRNA and gene silencing efficacy. A fluorescently labeled siRNA (FAM-siRNA-2) and an effective non-labeled siRNA (siRNA-4) targeting a luciferase fusion gene were individually diluted and transfected into HEK293 cells, together with the target reporter vector. To keep the siRNA concentration constant, an irrelevant siRNA was added into the dilutions. A: Distributions of the labeled siRNA 4 h after the transfections. B: Granule-aggregated siRNA was quantified by measuring the fluorescence intensity of the siRNA-aggregating granules, and was compared with the gene silencing efficacy of the unlabeled siRNA.
Fig.3  Aggregation of various exogenous nucleic acids in the same cytoplasmic granules. Single-stranded DNA oligonucleotide (FAM-ssDNA-1), double-stranded DNA oligonucleotide (FAM-dsDNA-1), single-stranded RNA oligonucleotide (FAM-ssRNA-1) as well as a plasmid DNA (YoYo-1-pDNA, an asymmetric cyanine dye from Molecular Probes) was individually transfected into HEK293 cells together with Alexa-647-siRNA-1. A: Distribution of the nucleic acids 4 h after the transfection, using laser confocal microscopy. Only merged images are shown. B: Prolonged observations for ssDNA transfection. C: Quantitative analysis of co-aggregation of siRNA with other nucleic acids. All the transfections were performed using Lipofectamine 2000 at an oligonucleotide concentration of 13 nmol/L, and the plasmid DNA was transfected at a concentration of 0.33 ng/μ L. Co-aggregation by chance was calculated as the control.
Fig.3  Aggregation of various exogenous nucleic acids in the same cytoplasmic granules. Single-stranded DNA oligonucleotide (FAM-ssDNA-1), double-stranded DNA oligonucleotide (FAM-dsDNA-1), single-stranded RNA oligonucleotide (FAM-ssRNA-1) as well as a plasmid DNA (YoYo-1-pDNA, an asymmetric cyanine dye from Molecular Probes) was individually transfected into HEK293 cells together with Alexa-647-siRNA-1. A: Distribution of the nucleic acids 4 h after the transfection, using laser confocal microscopy. Only merged images are shown. B: Prolonged observations for ssDNA transfection. C: Quantitative analysis of co-aggregation of siRNA with other nucleic acids. All the transfections were performed using Lipofectamine 2000 at an oligonucleotide concentration of 13 nmol/L, and the plasmid DNA was transfected at a concentration of 0.33 ng/μ L. Co-aggregation by chance was calculated as the control.
Fig.4  Formation of siRNA-aggregating granules is triggered by nucleic acid transfection. Time-lapse microscopy imaging was used to study the formation and cellular trafficking of the siRNA-aggregating granules, following transfection of a fluorescently labeled siRNA (Alexa-647-siRNA-1). A: Formation of the transfection granules. Distribution of the siRNA at different time points after transfection, showing the assembly process of the transfection granules. B: Time-lapse images showing the cellular trafficking of the granules. The time interval between frames is 1 min. The white arrows indicate a static transfection granule during the observation, while the black arrows indicate an actively trafficking granule. The size and fluorescence intensity of the granules remained constant during the observations. C: Two siRNAs labeled by different fluorophores, Alexa-647 (siRNA-1) or FAM (siRNA-3), were co-transfected or sequentially transfected into HEK293 cells. In sequential transfection, the second siRNA was transfected 2 h after the first one. Distributions of the siRNAs 4 h after the co-transfection or the second transfection in sequential transfection. All the transfections were performed with Lipofectamine 2000 at a concentration of 13 nmol/L.
Fig.4  Formation of siRNA-aggregating granules is triggered by nucleic acid transfection. Time-lapse microscopy imaging was used to study the formation and cellular trafficking of the siRNA-aggregating granules, following transfection of a fluorescently labeled siRNA (Alexa-647-siRNA-1). A: Formation of the transfection granules. Distribution of the siRNA at different time points after transfection, showing the assembly process of the transfection granules. B: Time-lapse images showing the cellular trafficking of the granules. The time interval between frames is 1 min. The white arrows indicate a static transfection granule during the observation, while the black arrows indicate an actively trafficking granule. The size and fluorescence intensity of the granules remained constant during the observations. C: Two siRNAs labeled by different fluorophores, Alexa-647 (siRNA-1) or FAM (siRNA-3), were co-transfected or sequentially transfected into HEK293 cells. In sequential transfection, the second siRNA was transfected 2 h after the first one. Distributions of the siRNAs 4 h after the co-transfection or the second transfection in sequential transfection. All the transfections were performed with Lipofectamine 2000 at a concentration of 13 nmol/L.
Fig.5  Granule-aggregation of plasmid DNA represses reporter gene expression. A: A fluorescently labeled siRNA (Alexa-647-siRNA-1) and a labeled plasmid DNA (a fusion luciferase reporter vector, YoYo-1) were co-transfected into HEK293 cells using INTERFERin, siPORT or Lipofectamine 2000. The distributions of siRNA and plasmid DNA 4 h after the transfections are shown. B: An effective siRNA (siRNA-4) targeting a luciferase fusion gene was co-transfected into HEK293 cells together with its target reporter vector using INTERFERin, siPORT or Lipofectamine 2000. The gene silencing efficacies were measured 24 h after the transfections by luciferase assay. The transfections were performed according to the respective product manual. The plasmid DNA was transfected at a concentration of 0.33 ng/μ L, and the siRNA was transfected at a concentration of 13 nmol/L.
Fig.5  Granule-aggregation of plasmid DNA represses reporter gene expression. A: A fluorescently labeled siRNA (Alexa-647-siRNA-1) and a labeled plasmid DNA (a fusion luciferase reporter vector, YoYo-1) were co-transfected into HEK293 cells using INTERFERin, siPORT or Lipofectamine 2000. The distributions of siRNA and plasmid DNA 4 h after the transfections are shown. B: An effective siRNA (siRNA-4) targeting a luciferase fusion gene was co-transfected into HEK293 cells together with its target reporter vector using INTERFERin, siPORT or Lipofectamine 2000. The gene silencing efficacies were measured 24 h after the transfections by luciferase assay. The transfections were performed according to the respective product manual. The plasmid DNA was transfected at a concentration of 0.33 ng/μ L, and the siRNA was transfected at a concentration of 13 nmol/L.
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