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, 9 (8), e103695
eCollection

Anti-PABPC1 Co-Immunoprecipitation for Examining the miRNAs Directly Targeting the 3'-UTR of EED mRNA

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Anti-PABPC1 Co-Immunoprecipitation for Examining the miRNAs Directly Targeting the 3'-UTR of EED mRNA

Yi Hu et al. PLoS One.

Abstract

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate post-transcriptional gene expression by base pairing with partially complementary sequences within target messenger RNAs (mRNAs). Although the target genes and the precise biological functions of individual miRNAs remain largely unknown, miRNAs have been implicated in diverse biological processes, including both normal and pathological states. As a single stranded mRNA can be directly targeted by multiple miRNAs, and as the target sites may exist in the 3'-untranslated region (UTR), 5'-UTR, or the coding regions, it is essential to develop an effective method to identify the full-scale miRNA regulatory pattern of each particular gene. In this study, we employed a biochemical approach to identify the miRNA profiles that regulate the expression of embryonic ectoderm development (EED) protein by using anti-PABPC1 ribonucleoprotein (RNP) co-immunoprecipitation (Co-IP). The full length EED mRNA was subcloned into an expression vector and transiently transfected into a Flag-PABPC1 stable expression cell line. Subsequent to cross-linking and an anti-Flag Co-IP, the miRNAs that directly targeted EED were identified. We found that the best time point to distinguish the positive miRNAs from the background was 18 hours after the plasmid transfection. As expected, the miRNAs that directly target EED were found to interact with EED mRNA through the miRNA-induced silencing complex (miRISC). Meanwhile, the EED mRNA was bound by Flag-PABPC1. This method depends on the integrity of the miRISC complex and achieves greater efficiency when ultraviolet irradiation is used for the process of cross-linking. By using anti-PABPC1 RIP, we identified EED to be a new target gene of miR-16; a finding further confirmed using a dual-luciferase assay. In summary, our data indicate that anti-PABPC1 RIP is a validated and direct biochemical method to provide data about specific miRNA-mRNA interactions, as well as global miRNA patterns regulating the mRNAs.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Schematic presentation of RNP immunoprecipitation.
PABPC1 participates in the process of post-transcriptional regulation of gene expression by miRNA. Therefore, we used anti-PABPC1 to identify the miRNAs that target EED mRNA.
Figure 2
Figure 2. Construction of a quality control system.
(A) A schematic diagram for the subcloning of the 3′-UTR of Lin28 and ERBB2 into the pGL3 vector. (B) HEK293T cells were co-transfected with Let-7b or miR-125a and the 3′-UTR of Lin28 or ERBB2 for the dual-luciferase assay. PRL-TK plasmid expressing the Renilla luciferase was used as a transfection control. The luciferase activity was detected 48 h after transfection, and the results were analyzed using the Student’s t-test. *P<0.05, **P<0.01.
Figure 3
Figure 3. Time course and efficiency of the anti-Flag PABPC1 RIP.
Plasmids containing the 3′-UTR of the wild type or mutant Lin28 (A) or ERBB2 (B) were transfected into the Flag-PABPC1 stable cell line. Anti-Flag PABPC1 RIP was used at four time points (12 h, 18 h, 24 h, and 36 h) after the transfection, and the total RNA in the cell lysate and precipitate was extracted using the TRIzol reagent. The miRNAs were detected by RT-qPCR and the results were analyzed using the Student’s t-test. P<0.05 was considered statistically significant. *P<0.05, **P<0.01.
Figure 4
Figure 4. The integrated RISC complex is needed for miRNA recruitment.
(A) Western blot analysis of the co-IPed products. Co-IPs were performed on cells transfected with the reporter plasmid LIN28 or LIN28-Del. As expected, the Flag-PABPC1 proteins were co-IPed with the anti-Flag antibody. Meanwhile, the anti-Flag antibody could not pull down the wild type PABPC1 in the HEK293T cell lysate. AGO2 and PAN2 were detected in the co-IPed products using anti-PAN2 and anti-AGO antibodies, respectively. RNase A was added to determine whether the co-IPed RISC-related components were affected by RNA degradation. In the RNase A-treated groups, the amounts of AGO2 and PAN2 were reduced, implying that the binding interactions between PABPC1 and AGO2 or PABPC1 and PAN2 are partially mRNA-dependent. (B) Knock down of endogenous AGO2 using siRNA. HEK293T cells were transfected with AGO2 siRNAs. At the end of the transfection (48 h), the cells were lysed and AGO2 expression was detected with a western blot. The knockdown effect was most effective in siAGO2-2. (C) Comparison of the miR-125a/Let-7b contents among the AGO2 knockdown groups and control groups. Cells were transfected with AGO2 siRNA. A scramble sequence and non-meaning short RNA was used as control. Cells were subsequently (after 48 h) divided into two dishes and transfected with the wild type or mutant LIN28 or ERBB2 plasmids, respectively. Anti-Flag co-IP was performed 18 h after the transfection and miR-125a and Let-7b were detected by RT-qPCR. The results were analyzed with the Student’s t-test and P<0.05 was considered statistically significant.
Figure 5
Figure 5. Anti-PABPC1 RNP immunoprecipitation for identifying miRNAs that target EED.
(A) Western blot analysis of co-IPed products. Cells were transfected with the EED expression vector, with empty pVAX1 as the control. Cells were treated with 150 mJ/cm2 UV (lanes 3, 4) or formaldehyde (FM) (lanes 5, 6) 18 h after transfection to cross-link the protein-RNA complex. The cells were then lysed and co-IPed. As expected, the FLAG-PABPC1 proteins were immunoprecipitated with the anti-FLAG antibody and the FLAG-PABPC1 protein contents were nearly the same in the non-cross-linking and UV cross-linking groups. However, the FLAG-PABPC1 amount was reduced in the lysate and precipitate of the formaldehyde treatment group compared to those of the non-cross-linking and UV cross-linking groups. (B) The EED mRNA levels were detected by RT-qPCR and the results were analyzed using the Student’s t-test. P<0.05 was considered statistically significant. (C) The levels of four selected miRNAs in the precipitate were detected by RT-qPCR. Results were analyzed by the Student’s t-test. *P<0.05, **P<0.01.
Figure 6
Figure 6. MiR-16 and miR-101 suppress EED expression through targeting to the 3′-UTR of EED.
(A) The predicted results of miRanda indicated that EED is targeted by miR-16 and miR-101, whereas the TargetScan online tools indicated that there could be a direct interaction between miR-16 and EED mRNA. (B) Confirmation of the relationship between EED and miR-101/miR-16. Cells were co-transfected with the miRNA mimic control, the miR-101 mimic, the miR-16 mimic, or the miR-181b mimic, and the Luc-EED for the dual-luciferase assay. PRL-TK containing Renilla luciferase was co-transfected with the 3′-UTR of EED for data normalization. *P<0.05, **P<0.01.
Figure 7
Figure 7. Identifying the target sites of miR-16 and miR-101 in the 3′-UTR of EED.
The predicted binding site of miR-16 and miR-101 in the 3′-UTR of EED was mutated (designated as Luc-EED-Mu1 and Luc-EED-Mu1, respectively). HEK293T cells were transfected with miR-16 or 101, and the mutant EED 3′-UTR reporter vectors separately, with pRL-TK as the transfection control. The luciferase activity was detected 48 h after transfection. Results were analyzed with the Student’s t-test. *P<0.05, **P<0.01.

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Grant support

This work was funded by grants from Natural Science Foundation of China (No. 81370720, No. 81301446). http://www.nsfc.gov.cn/Portal0/default166.htm. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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