A Study On The Regulatory Mechanism Of RNA Polymerase Ⅲ-directed Gene Transcription Mediated By RPS6KA2

BackgroundEukaryoticRNA polymerase Ⅲ(Pol Ⅲ)is responsible for synthesizing tRNA,5S rRNA,7SLRNA,U6 SnRNA,and other noncodingRNAs in cells.Pol Ⅲ products play an important role in ribosome assembly,protein synthesis and transportation,gene transcription regulation,metabolism,and cell growth.Dysregulation of gene transcription directed by Pol Ⅲ closely correlates with tumorigenesis and cell proliferation.Ribosomal protein S6 kinase A2(RPS6KA2,alias RSK3)is a member of the serine/threonine kinase RSK(ribosomal S6 kinase)family.This kinase contains two distinct kinase catalytic domains and is involved in the regulation of various physiological and biochemical activities such as cell growth and differentiation by phosphorylating different substrates.Previous results in my laboratory have shown that RPS6KA2 promotes gene transcription directed by Pol Ⅲ,but the regulatory mechanism in this event is unclear.In this project,various molecular and biological approaches were used to study and uncover the regulatory mechanism of Pol Ⅲ gene transcription mediated by RPS6KA2.MethodsRPS6KA2 shRNA and RPS6KA2-m Cherry lentiviral expression vectors and the packaged plasmids p H1 and p H2 were used to transfect 293 T cells,where lentiviruses were packaged.Subsequently,the obtained lentiviruses were used for the transduction of He La and Hep G2 cells to establish the stable cell lines with RPS6KA2 knockdown or overexpression.Western blot and RT-q PCR were used to detect the expression of RPS6KA2 and Pol Ⅲ products in stable cell lines,respectively.Cell counting,Ed U,colony formation,and tumor formation assays with nude mice were used to analyze the effect of RPS6KA2 expression changes on cell proliferative activity.Chromatin immunoprecipitation(Ch IP),Western blot,and RPS6KA2 and p21 double knockdown experiments were used to investigate the regulatory mechanisms of Pol Ⅲ gene transcription mediated by RPS6KA2.ResultsRPS6KA2 knockdown inhibited Pol Ⅲ-directed gene transcription,while RPS6KA2 overexpression promoted the expression of Pol Ⅲ products;indicating that RPS6 KA acts as an activator in Pol Ⅲ transcription.The results from cell proliferation assays and tumor formation assays showed that RPS6KA2 promotes tumor cell proliferation in vivo and in vitro.The effect of Pol Ⅲ inhibitors on RPS6KA2 overexpressed cell lines revealed that the activation of cell proliferation mediated by RPS6KA2 was affected by Pol Ⅲ transcription levels.Ch IP-q PCR results showed that RPS6KA2 knockdown reduced the binding of Pol Ⅲ transcription machinery factors to Pol Ⅲ target gene loci.Western blot results showed that RPS6KA2 was able to inhibit the expression of cyclin-dependent kinase inhibitor p21,but did not significantly affect the expression of most other proteins related to Pol Ⅲ transcriptional regulation,alternatively,RPS6KA2 differentially regulates some of the detected proteins.After establishing stable cell lines with knockdown of both RPS6KA2 and p21 and the cell line with p21 silencing,RT-q PCR was used to analyze Pol Ⅲ products;the results showed that p21 is not only involved in the regulation of Pol Ⅲ transcription mediated by RPS6KA2,but also independently regulates Pol Ⅲ transcription.Transfection of miRNA mimimics targeting p21 mRNA reduced p21 expression and increased the expression of Pol Ⅲ products.The results fromRNA immunoprecipitation and RT-q PCR suggest that RPS6KA2 can bind to mi R-95-3p in the cytoplasm.ConclusionRPS6KA2 can bind to mi R-95-3p and affect the expression or stability of mi R-95-3p.Upregulation of mi R-95-3p expression inhibits p21 expression.The inhibition of p21 expression leads to increased expression of TBP and GTF3C2,thereby promoting Pol Ⅲ-directed gene transcription.Thus,RPS6KA2 activates Pol Ⅲ-directed transcription by regulating the mi R-95-3p-p21-TBP/GTF3C2 pathway.These findings provide new insights into the mechanisms of gene transcription directed by Pol Ⅲ.