Investigating The Biological Functions Of RNA Polymerase Ⅰ Subunits RPA43 And RPA49 In Human Cells

RNA polymerase Ⅰ(Pol Ⅰ)synthesizes the majority of r RNA in eukaryotic cells,its products are closely correlated with protein synthesis and cell growth.Both RPA43 and RPA49 are Pol Ⅰ-specific subunits.Previous studies on the role of RPA43 in Pol Ⅰ transcriptional regulation are mostly focused on yeast,and there is very few study in mammals.The heterodimer formed by RPA49 and RPA34 is essential for Pol Ⅰ transcription,but their roles in Pol Ⅰ transcription is not yet clear.Therefore,in this project,we focused on the RPA43 and RPA49 proteins and investigated the role of RPA43 and RPA49 in Pol Ⅰ transcriptional regulation and explored the possible regulatory mechanisms underlying these processes.In order to achieve our research objectives,we first established HeLa and 293 T cell lines stably expressing RPA43 sh RNA or RPA49 sh RNA.RT-q PCR was used to analyze the expression of Pol Ⅰ transcripts in the cell lines with RPA43 or RPA49 knockdown.The results showed that either RPA43 or RPA49 knockdown could effectively inhibit the expression of Pol Ⅰ products.To verify this discovery,we subsequently established the stable cell lines expressing m Cherry-RPA43 or m Cherry-RPA49.RT-q PCR was performed using the stable cell lines with RPA43 or RPA49 overexpression.The results showed that overexpression of RPA49 was able to promote the expression of Pol Ⅰ products,whereas RPA43 showed cell type-specific regualtion for Pol Ⅰ transcription.The overexpression of RPA43 promoted Pol Ⅰ transcription in 293 T cells,while overexpression of RPA43 inhibited Pol Ⅰ transcription in HeLa cells.Previous studies have shown that Pol Ⅰ-directed gene transcription is closely correlated with cell growth and proliferation.The results of cell proliferation analysis by CCK8 and Ed U cell proliferation assays showed that RPA49 knockdown inhibited cell proliferation,while overexpression promoted cell proliferation.However,RPA43 also showed cell type-specific regualtion for cell proliferation.In 293 T cells,RPA43 knockdown inhibited cell proliferation,and overexpression promoted cell proliferation;whlie in HeLa cells,RPA43 knockdown inhibited cell proliferation,and overexpression also inhibited cell proliferation.Since Pol Ⅰ and Pol Ⅲ transcription are coordinately modulated in eukaryotic cells,we have also detected expression levels of Pol Ⅲ transcription products through RT-q PCR.The results showed that RPA49 knockdown repressed expression of Pol Ⅲ products,whereas its overexpression enhanced expression of Pol Ⅲ products.Knockdown of RPA43 in293 T cells inhibited expression of Pol Ⅲ products.In contrast,RPA43 overexpression stimulated expression of Pol Ⅲ products.However,in HeLa cells,knockdown of RPA43 activated expression of Pol Ⅲ products,while its overexpression had no effect on expression of some genes transcribed by Pol Ⅲ.Since cell proliferation is also influenced by Pol Ⅲ transcription level,the above results suggest that RPA43 and RPA49 possibly regulate cell proliferation by controlling Pol Ⅰ and Pol Ⅲ transcription.In order to explore the molecular mechanism that RPA49 regulated Pol Ⅰ gene transcription,Western blot was used to detect expression of transcription factors related to Pol Ⅰ transcription in the cell lines with RPA49 knockdown or overexpression.The results showed that RPA49 had a positive regulatory effect on expression of several transcription factors,including RRN3,UBF1,and TBP.These results give us a better understanding of the biological functions of Pol Ⅰ subunits RPA43 and RPA49,and lay a basis for diseccting the mechanism of Pol Ⅰ transcription regulation in the future work.