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. 2018 Jan 2;15(1):55-61.
doi: 10.1080/15476286.2017.1391441. Epub 2017 Nov 9.

Comparing Two Approaches of miR-34a Target Identification, biotinylated-miRNA Pulldown vs miRNA Overexpression

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Free PMC article

Comparing Two Approaches of miR-34a Target Identification, biotinylated-miRNA Pulldown vs miRNA Overexpression

Hassaan Mehboob Awan et al. RNA Biol. .
Free PMC article

Abstract

microRNAs (miRNAs) are critical regulators of gene expression. For elucidating functional roles of miRNAs, it is critical to identify their direct targets. There are debates about whether pulldown of biotinylated miRNA mimics can be used to identify miRNA targets or not. Here we show that biotin-labelled miR-34a can be loaded to AGO2, and AGO2 immunoprecipitation can pulldown biotinylated miR-34a (Bio-miR pulldown). RNA-sequencing (RNA-seq) of the Bio-miR pulldown RNAs efficiently identified miR-34a mRNA targets, which could be verified with luciferase assays. In contrast to the approach of Bio-miR pulldown, RNA-seq of miR-34a overexpression samples had limited value in identifying direct targets of miR-34a. It seems that pulldown of 3'-Biotin-tagged miRNA can identify bona fide microRNA targets at least for miR-34a.

Keywords: Argonaut 2; Biotinylated miRNA; RNA pull down; RNA-seq; microRNA targets.

Figures

Figure 1.
Figure 1.
Biotin-labelled miRNA co-immunoprecipitates with AGO2: (A) Schematic of AGO2 immunoprecipitation (IP) and biotin-labelled miRNA detection. (B) AGO2 IP of Bio-miR-Scr and Bio-miR-34a transfected HEK293T cells. Western blots of AGO2 showing successful IP, and Actin was used as a negative control. (C) Bio-miR-Scr and Bio-miR-34a both co-IPed with AGO2 but not with IgG. Arrow points towards biotinylated miRNA (bands were probed with streptavidin-HRP).
Figure 2.
Figure 2.
AGO2 co-immunoprecipitates with biotin-labelled miRNA: (A) RNA pulldown of Bio-miR-Scr and Bio-miR-34a in HEK293T cells. Bands were probed with streptavidin-HRP. (B) AGO2 co-pulled down with both Bio-miR-Scr and Bio-miR-34a. Western blots of AGO2 were performed, and β-tubulin was used as a negative control.
Figure 3.
Figure 3.
RNA-Sequencing data analyses: (A) Schematic of sample preparation for RNA-seq. (B) Total number of pulled down mRNAs and lncRNAs from Bio-miR-34a pull down and RNA-seq. (C) Number of pulled down mRNAs and lncRNAs after normalizing with Bio-miR-scramble pulldown. (D) Number of mRNAs and lncRNAs downregulated after miR-34a overexpression. (E) Comparison of pulled down and downregulated mRNAs in search of common targets. (F) Comparison of pulled down targets with downregulated mRNAs due to guide strand (miR-34a-5p) and passenger strand (miR-34a-3p). (G) Pulled down targets with 3′ UTR seed match compared with downregulated mRNAs due to guide strand (miR-34a-5p) and passenger strand (miR-34a-3p). (H) Cumulative distribution plots for Bio- miR-34a pull down targets (n = 139, p < 0.05) and scramble control pull down targets (randomly chosen 103 genes, p>0.05) showing percentage of genes with average log2 (miR-34a mimic/scramble control) indicated on the x-axis.
Figure 4.
Figure 4.
Validation of potential miR-34a targets: Firefly luciferase activity of; (A) randomly chosen pulldown targets, (B) randomly chosen downregulated mRNAs in RNA-seq data after miR-34a overexpression and, (C) overlapped targets, in HEK293T cells relative to Renilla luciferase. Data represents values from triplicates. P values were generated using two-tailed Student's t-test. ns, not significant; **P < 0.01; ***P < 0.001. Error bars represents S.D.

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