In pre-initiation complexes, RNA helicase A interacts with -actin and acts as a bridging factor linking nuclear actin with RNA polymerase II (Pol II). In addition, -actin participates in Pol II-dependent transcription elongation by interacting with the positive transcription elongation factor Cdk9. However, many relationships between -actin and Pol II remain to be identified. In an interleukin 6 (IL-6)-induced p21 expression model, we demonstrated that -actin knockdown reduced p21 expression. Immunofluorescence analysis showed that the colocalization of -actin and Pol II increased significantly in cells treated with IL-6. It is known that the Rpb5, Rpb6 and Rpb7 subunits are located at the surface of the enzyme. We next constructed recombinant pcDNA-HA-Rpb5, pcDNA-HA-Rpb6 and pcDNA-HA-Rpb7 plasmids and expressed the three polymerase II subunits in HepG2 cells. We found that -actin could be immunoprecipitated with HA-Rpb5 and HA-Rpb7. A Glutathione-S-transferase pull-down assay revealed that -actin was associated with Rpb5 and Rpb7 in vitro. Furthermore, overexpression of Rpb5 and Rpb7 in cells reduced p21 expression significantly, suggesting that Rpb5 and Rpb7 competitively interact with -actin. This study shows that -actin associates with Pol II subunits through direct protein-protein interactions and provides fundamental insight into Pol II transcriptional regulation.Abbreviations: DAPI: 4,6-diamidino-2-phenylindole; FBS: fetal bovine serum; HAT: histone acetyltransferase; HDAC: histone deacetylase; GST: Glutathione-S-transferase; IL-6: interleukin 6; PICs: pre-initiation complexes; Pol II: RNA polymerase II; RHA: RNA helicase A; siRNA: small interfering RNA
[1]Jilin Univ, China Japan Union Hosp, Changchun, Peoples R China.
[2]Changchun Normal Univ, Dept Biosci, Changchun, Peoples R China.
[3]Northeast Normal Univ, Inst Cytol & Genet, Sch Life Sci, Key Lab Mol Epigenet MOE, Changchun, Peoples R China.
(8.0+极速模式)