Cellular miRNAs and AGO proteins are associated with RNA Pol II core transcription machinery. (A) Autoradiogram of 32P-labeled RNA associated with human RNA Pol II purified by RNA chromatin immunoprecipitation (RNA-ChIP). The PBMCs were isolated from healthy individuals and activated with anti-CD3 (1 μg/mL) and anti-CD28 (5 μg/mL) antibodies for 48 h. RNA-ChIP assay was then performed. The chromatin-associated RNA was extracted and radiolabeled. These data represent three independent experiments. (B) Identification of RNA Pol II-associated miRNAs with miRNA expression microarray. The table only shows the detected miRNAs. qRT-PCR quantification of the miRNAs associated with RNA Pol II (C) and TBP (D) with or without DNase treatment. The enriched miRNAs were determined by real-time qRT-PCR and normalized to that of IgG. P-values were calculated using the two tailed unpaired Student's t-test with equal variances, n = 3 biological replicates. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. Co-immunoprecipitation (co-IP) assay to detect the association of HA-tagged Ago proteins with RNA Pol II (E) or TBP (F). HA-tagged Ago1 and Ago2 constructs were transfected into HEK293T cells. After 48 h, cell lysates were immunoprecipitated with anti-HA antibody, and subsequently detected with anti-RNA Pol II or anti-TBP antibody. (*) Nonspecific band. These data represent at least three independent experiments.
