Involvement of argonaute proteins in gene silencing and activation by RNAs complementary to a non-coding transcript at the progesterone receptor promoter - PubMed (original) (raw)
Involvement of argonaute proteins in gene silencing and activation by RNAs complementary to a non-coding transcript at the progesterone receptor promoter
Yongjun Chu et al. Nucleic Acids Res. 2010 Nov.
Abstract
Double-stranded RNAs that are complementary to non-coding transcripts at gene promoters can activate or inhibit gene expression in mammalian cells. Understanding the mechanism for modulating gene expression by promoter-targeted antigene RNAs (agRNAs) will require identification of the proteins involved in recognition. Previous reports have implicated argonaute (AGO) proteins, but identifications have differed with involvement of AGO1, AGO2, or both AGO1 and AGO2 being reported by different studies. The roles of AGO3 and AGO4 have not been investigated. Here, we examine the role of AGO 1-4 in gene silencing and activation of the progesterone receptor (PR) gene. Expression of AGO2 is necessary for efficient gene silencing or activation and AGO2 is recruited to the non-coding transcript that overlaps the promoter during both gene silencing and activation. Expression of AGO1, AGO3 and AGO4 are not necessary for gene silencing or activation nor are AGO1, AGO3, or AGO4 recruited to the target non-coding transcript during gene activation. These data indicate that AGO2 is the primary AGO variant involved in modulating expression of PR by agRNAs.
Figures
Figure 1.
Scheme showing PR gene promoter structure and the effect of promoter targeted agRNAs on the recruitment of RNAP2 to the transcription start site. (A) The regions targeted by representative agRNA duplexes. Relative to PR-B transcription starting site (TSS), PR-26, PR-11 and PR-9 target −26 to −7, −11 to +8 and −9 to +10 regions, respectively. (B) The non-coding 5′-antisense transcript AT2 starts within the coding region of PR in both T47D and MCF7 cells. ChIP assay evaluates the recruitment of RNAP2 in (C) T47D cells treated by agRNA PR-9; (D) MCF7 cells treated by agRNA PR-11. MM: negative control RNA duplex. Mouse IgG was used as a negative control antibody. The experiments were repeated at least three times. ***P < 0.005 as compared to cells treated with a mismatched RNA (MM). _P_-values were calculated using the two tailed unpaired Student’s _t_-test with equal variances. All error bars represent standard deviation.
Figure 2.
AGO expression and silencing. qPCR analysis of relative expression levels of AGO1–4 in human breast cancer cell lines, (A) T47D and (B) MCF7. Effect of silencing (C) AGO1, (D) AGO2, (E) AGO3 and (F) AGO4 on expression of other AGO variants (using 25 nM siRNA). All data were normalized to GAPDH. Error bars represent standard deviations, calculated from four independent experiments. ***P < 0.005, **P < 0.01, *P < 0.05 as compared to cells treated with a mismatched RNA (MM). _P_-values were calculated using the two tailed unpaired Student’s _t_-test with equal variances.
Figure 3.
Effect of inhibiting AGO1–4 or dicer on expression of PR mRNA, PR antisense transcript (as-PR), and PR protein. (A) qPCR analysis of PR and as-PR after silencing expression of AGO1–4 or dicer by siRNA in T47D cells. (B) qRT-PCR analysis of PR and as-PR after a repression of AGO1–4 or dicer by siRNA in MCF7 cells. (C) Western analysis of PR protein after inhibiting expression of AGO1–4 or dicer in T47D cells. (D) Western analysis of PR protein following inhibition of AGO1–4 or dicer in MCF7 cells. PR-9 and PR-11: positive controls. MM: negative control. The final concentration for all used RNAs was 25 nM. qPCR data was normalized relative to GAPDH. Error bars represent standard deviations, calculated from four independent experiments. ***P < 0.005, **P < 0.01, *P < 0.05 as compared to cells treated with a mismatched RNA (MM). _P-_values were calculated using the two tailed unpaired Student’s _t_-test with equal variances.
Figure 4.
Effect of reduced expression of AGO1, AGO2, AGO3, or AGO4 (siRNAs, 25 nM) on silencing by PR-9 (25 nM) in T47D cells. qPCR analysis of PR after a double transfection assay with (A) siAGO1, (B) siAGO2, (C) siAGO3, (D) siAGO4 being added first and PR9 added second (in 72 h). Western analysis of PR protein levels after a double transfection assay with (E) siAGO1, (F) siAGO2, (G) siAGO3, (H) siAGO4 being added first and PR-9 added second (in 72 h). Transfection combinations: MM/PR-9 represents that the first transfection is MM, a control RNA duplex and the second is PR-9, etc. All data were normalized to GAPDH. Error bars represent standard deviations, calculated from three independent experiments. ***P < 0.005 as compared to cells treated with a mismatched RNA (MM). _P_-values were calculated using the two tailed unpaired Student’s _t_-test with equal variances.
Figure 5.
Effect of inhibiting AGO1, AGO2, AGO3 or AGO4 gene expression on activation of PR expression by PR-11 (25 nM) in MCF7 cells. qPCR analysis of PR after a double transfection assay with (A) siAGO1, (B) siAGO2, (C) siAGO3, (D) siAGO4 being added first (Day 0) and PR-11 added second (after 72 h). Western analysis of PR after a double transfection assay with (E) siAGO1, (F) siAGO2, (G) siAGO3, (H) siAGO4 being added first (Day 0) and PR-11 added second (after 72 h). Transfection combinations: MM/PR-11 represents that the first transfection is MM, a control RNA duplex and the second is PR-11, etc. All data were normalized to GAPDH for q-PCR. Error bars represent standard deviations, calculated from three independent experiments. ***P < 0.005 as compared to cells treated with a mismatched RNA (MM). _P_-values were calculated using the two tailed unpaired Student’s _t_-test with equal variances.
Figure 6.
Association of AGO1–4 with the PR antisense transcript upon addition of PR-9 or PR-11 determined by RIP. (A) Western analysis of AGO1, AGO2, AGO3 and AGO4 proteins in whole cell and nuclear fractions from T47D cells. 40 µg of protein was loaded in each lane. WC: whole cell fraction; Nuc: nuclear fraction. (B) The nuclear distribution of PR mRNA, GAPDH mRNA and PR pre-mRNA in MCF7 cells. Data is represented as percent ratio of amplified transcript in nuclear to whole cell extracts. SN 7SK was used as a nuclear internal control. (C) RIP examining association of AGO1, AGO2, AGO3 and AGO4 with the PR antisense transcript in T47D cells treated with PR-9. (D) RIP examining association of AGO1, AGO2, AGO3 and AGO4 with the PR antisense transcript in MCF7 cells treated with PR-11. MM: negative control RNA duplex. IgG: negative control antibody. Input: nuclear extract prior to treatment with antibody. Input NR: input sample with no reverse transcriptase treated. The above data is the representative data set from three independent experiments.
Figure 7.
RIP examining the association of Flag/HA tagged AGO proteins with the PR antisense transcript. MCF7 cells were transfected with Flag/HA-Ago plasmid first and then PR-11 in 48 h (final 25 nM). (A) Flag/HA-AGO1, (B) Flag/HA-AGO2, (C) Flag/HA-AGO3 and (D) Flag/HA-AGO4, were examined. The transfection protocol is described in the ‘Materials and Methods’ section. P-A1: Flag/HA-AGO1 plasmid, etc. MM: negative control RNA duplex. IgG: negative control antibody. Input: nuclear extract prior to treatment with antibody. Input NR: input sample with no reverse transcriptase treated. The above data is the representative data set from three independent experiments.
References
- Siomi H, Siomi MC. On the road to reading the RNA-interference code. Nature. 2009;457:396–404. - PubMed
- Morris KV, Chan SW, Jacobsen SE, Looney DJ. Small interfering RNA-induced transcriptional gene silencing in human cells. Science. 2004;305:1289–1292. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Research Materials