Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: MCPIP1 overexpression inhibits de novo KSHV infection. (A) Schematic of KSHV infection timeline following MCPIP1 overexpression. 293T-GFP and 293T-GFP-MCPIP1 cells were stimulated with 10 ng/ml doxycycline (DOX) or DMSO (unstimulated control) for 24 h prior to KSHV infection (before infection), 6h post infection (after infection), or both prior to and following infection (before and after infection). At 96h post KSHV infection, MCPIP1 expression (C and D), viral genome copies (E), and viral gene expression (F and G) were analysed. (B) 293T-GFP and 293T-GFP-MCPIP1 cells were exposed to 10 ng/ml DOX for 0, 24h, and 48h. The levels of MCPIP1 transcripts were analysed by qPCR. Results are shown relative to GAPDH and normalized to DMSO treated control. (C) Levels of MCPIP1 transcripts was analysed by qPCR. Results are shown relative to GAPDH and normalized to unstimulated control. (D and G) MCPIP1 was induced before and after infection followed by KSHV infection in 293T-GFP-MCPIP1 cells. (D) Levels of MCPIP1 protein were analysed. β-actin (ACT) was used as a loading control. (E) Viral genome copies were measured by qPCR using primers specific to the KSHV ORF73(LANA) gene. Results are shown relative to BRCA1 and normalized to unstimulated control (F) Levels of ORF73(LANA), ORF50(RTA), ORF6, ORF60, ORF34 and K8.1 transcripts were analysed by qPCR. Results are shown relative to GAPDH and normalized to unstimulated control (G) Level of LANA protein was measured by immunoblot in nuclear and cytoplasm extracts from cells. Nuclear and cytoplasmic fractions were determined by Lamin B (nuclear marker) and HSP90 (cytoplasmic marker). All results are represented standard error of the mean from independent experiments, N = 3–4 and statistical analysis was done with a Student's t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques: Over Expression, Infection, Expressing, Western Blot, Marker

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: MCPIP1 knockdown enhances viral genome accumulation. (A) Schematic of timeline of KSHV infection in HUVEC following MCPIP1 knockdown. HUVECs were stimulated with IL-1β for 2 h following transfection with negative control Locked Nucleic Acid (LNA) gapmer or LNA gapmers targeting MCPIP1 for repression, subsequently the cells were infected by KSHV. At 6 h post infection, IL-1β was treated until samples were collected. At 72 h post infection, cells were harvested. (B) At 26 h post-transfection, efficiency of MCPIP1 knockdown in HUVECs was determined by qPCR. Results are shown relative to GAPDH and normalized to negative gapmer control (C) At 98 h post transfection, MCPIP1 expression was analysed by qPCR. Results are shown relative to GAPDH and normalized to negative gapmer control (D) Viral genome copies were measured by qPCR using primers specific to the ORF73(LANA). Results are shown relative to BRCA1 and normalized to negative gapmer control. All results are represented standard error of the mean from independent experiments, N = 3 and statistical analysis was done with a Student's t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques: Infection, Transfection, Negative Control, Expressing

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: Determining the correlation of MCPIP1 cleavage efficiency and binding affinity. (A) Cleavage efficiency was evaluated by in vitro cleavage assay. Fluorescently labelled pre-miRNAs were incubated with recombinant MCPIP1, before separation on a denaturing urea gel. The percent of full-length pre-miRNAs remaining was quantified in the accompanying graph. Size markers (left) as well as full length and cleaved RNA products (right) are indicated. (B and C) Binding affinity was determined by electrophoretic mobility shift assay (EMSA). (B) Fluorescently labelled pre-miRNAs at 12 nM were incubated with recombinant MCPIP1 D141N (RNase dead mutant) at 0, 100, 200, 400, 600 and 800 nM (lanes 1- 6, respectively), before separation on a 0.8% agarose gel. The unbound RNA and MCPIP1 D141N bound RNA complexes are marked on the left. (C) The fraction of bound RNA was plotted against MCPIP1 concentration to calculate the equilibrium dissociation constant, Kd. (D) The relation between Kd and the percent of full-length pre-miRNAs were plotted. Correlation was analysed using Pearson's correlation coefficient (r) and linear regression line is overlaid. A P-value of <0.05 was considered significant. Each point represents results obtained from each pre-miRNA. All results are represented as means ± SD of data from independent experiments, N = 3.

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques: Binding Assay, In Vitro, Cleavage Assay, Incubation, Recombinant, Electrophoretic Mobility Shift Assay, Mutagenesis, Agarose Gel Electrophoresis, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: Comparison of binding properties and cleavage efficiency of wild type and M5 base motif mutant pre-miRNAs with MCPIP1. (A) Predicted secondary structure of kshv-mir-K12–3 WT and its M5 base motif mutants (left) and hsa-let-7c WT and its M5 base motif mutant (right) based on mfold structure predictions. M5 base motif is highlighted. Cleavage efficiency was evaluated by in vitro cleavage assay. Fluorescently labelled pre-miRNAs were incubated with recombinant MCPIP1, before separation on a denaturing urea gel. The percent of full-length pre-miRNAs remaining was quantified in the accompanying graph. Size markers as well as full length and cleaved RNA products are indicated. WT, wild type and mut, mutant. (B) Binding affinity was determined by EMSA. Fluorescently labelled kshv-mir-K12–3 WT and its M5 base motif mutants (up) or fluorescently labelled hsa-let-7c WT and its M5 base motif mutant (low) were incubated with recombinant MCPIP1 D141N at 0, 100, 200, 400, 600 and 800nM (lanes 1–6) or at 0, 25, 50, 100, 200, 400, 600 and 800nM (lanes 1–8) respectively, before separation on a 0.8% agarose gel. The unbound RNA and MCPIP1 D141N bound RNA complexes are marked on the left. The fraction of bound RNA was plotted against MCPIP1 concentration in nM to calculate the equilibrium dissociation constant, Kd. (C) The relation between Kd and the percent of full-length pre-miRNAs were plotted. Correlation was analysed using Pearson's correlation coefficient (r) and linear regression line is overlaid. A P-value of < 0.05 was considered significant. Each point represents results obtained from each pre-miRNA. All results are represented as means ± SD of data from independent experiments, N = 3–4.

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques: Binding Assay, Mutagenesis, In Vitro, Cleavage Assay, Incubation, Recombinant, Agarose Gel Electrophoresis, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: Comparison of cleavage efficiency of wild type and M5 base motif introduced mutant kshv-mir-K12–6 with MCPIP1. (A) Predicted secondary structure of kshv-mir-K12–6 WT and its M5 base motif introduced mutant based on mfold structure predictions. (B) Cleavage efficiency was evaluated by in vitro cleavage assay. Fluorescently labelled kshv-mir-K12–6 and its mutant were incubated with recombinant WT MCPIP1 or MCPIP1 D141N, before electrophoresis on a denaturing urea gel. Size markers as well as full length and cleaved RNA products are indicated. (C) Each replicate of full-length pre-miRNAs remaining following MCPIP1 treatment was quantified in the graph, corresponding to lanes 2 and 5 in panel (B).

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques: Mutagenesis, In Vitro, Cleavage Assay, Incubation, Recombinant, Electrophoresis

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: Cleavage efficiency of Dicer1 3′ UTR regions was evaluated by in vitro cleavage assays. Fluorescently labelled Dicer1 3′ UTR fragment RNAs were incubated with recombinant MCPIP1 or MCPIP1 D141N, before electrophoresis on a denaturing urea gel. The percent of full-length RNAs remaining for Dicer1 3′ UTR region1 and its YRY mutant (up) and Dicer1 3′ UTR region2 and its YRY mutant were quantified in the accompanying graph. Size markers(left) as well as full length and cleaved RNA products are indicated. All results are represented as means ± SD of data from independent experiments, N = 3.

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques: In Vitro, Incubation, Recombinant, Electrophoresis, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: Analysis of terminal loop sequences of KSHV pre-miRNAs. (A) Loop sequences of KSHV pre-miRNAs for the negative set (less degraded by MCPIP1) and the positive set (more degraded by MCPIP1) that used in MEME program for motif discovery. (B) M5 base motif was found by the MEME program within the terminal loops of KSHV pre-miRNAs that were well degraded by MCPIP1.

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques:

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: Examination of Dicer expression following MCPIP1 overexpression. 293T-GFP and 293T-GFP-MCPIP1 cells were exposed to 10 ng/ml DOX for 0, 24 and 48 h (A) or for 48 h (B). (A) The levels of Dicer1 transcripts were measured by qPCR. Results are shown relative to GAPDH and normalized to DMSO treated control. (B) Levels of Dicer and GFP-MCPIP1 protein were examined by immunoblot. Results are shown relative to actin and normalized to unstimulated control. All results are represented as means ± SD of data from independent experiments, N = 3 and statistical analysis was done with a Student's t test. *P < 0.05.

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques: Expressing, Over Expression, Western Blot

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: A schematic model depicting the MCPIP1 mediated inhibition of KSHV infection. Upon KSHV infection, positive miRNA biogenesis factors, Dicer expression levels are enhanced while the expression of the negative miRNA biogenesis factor, MCPIP1, is suppressed to provide favourable environment for viral miRNA biogenesis. However, the human host induces the expression of inflammatory cytokines upon infection, which upregulates MCPIP1 expression. MCPIP1 expression inhibits KSHV miRNA biogenesis by directly degrading KSHV miRNAs and by suppressing the expression of a positive miRNA biogenesis factor, Dicer. Furthermore, MCPIP1 inhibits KSHV infection by suppressing the expression of Dicer that positively regulates KSHV infection.

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques: Inhibition, Infection, Expressing

Journal: eLife

Article Title: Overcoming the cytoplasmic retention of GDOWN1 modulates global transcription and facilitates stress adaptation

doi: 10.7554/eLife.79116

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , Doxycycline (Dox) , Biogems, California, USA , Cat# 2431450 , .

Techniques: Recombinant, Plasmid Preparation, Bimolecular Fluorescence Complementation Assay, Expressing, CRISPR, Generated, Marker, Purification, Imaging, Software, Microscopy, Methylation, Transfection