Investigating The Biological Functions Of RNA Polymerase ? Subunits RPA 12 And RPA34 In Human Cells

Eukaryotic RNA polymerase ?(Pol?)is a multi-subunit complex that is responsible for the synthesis of 45 S precursor ribosomal RNA(pre-r RNA).Both RPA12(DNAdirected RNA polymerase ? subunit RPA12)and RPA34(DNA-directed RNA polymerase ? subunit RPA34)are unique subunits of RNA polymerase ?,RPA12 is a protein containing two potential zinc-binding motifs.Previous studies have shown that yeast RPA12 plays a role in the synthesis of pre-r RNA and lipid metabolism.However,the biological functions of RPA12 in human cells are rarely studied;RPA34 is also referred to as ASE-1,CD3 EAP,CAST,etc.It was first discovered to exist in the nucleolar organizer regions of the split chromosomes and participates in the signal transduction of the T cell receptor complexes.But the role of RPA34 in human cells remains unclear.In order to complete the research on this subject,we cloned RPA12 sh RNA lentiviral expression vector,RPA34 sh RNA lentiviral expression vector,m Cherry-RPA12 lentiviral expression vector and m Cherry-RPA34 lentiviral expression vector.They were cotransfected with lentiviral packaging plasmids into 293 T cells and He La cells at a fixed ratio,and established RPA12 knockdown stable cell line,RPA34 knockdown stable cell line,RPA12 overexpression stable cell line and RPA34 overexpression stable cell line.We used RT-q PCR to detect the expression level of Pol?-directed gene transcription products in the above cell lines.The experimental results obtained in this subject are as follows:(1)Knockdown of RPA12 could significantly inhibit the expression of the gene transcription products directed by Pol?;similarly,knockdown of RPA34 was also able to inhibit the transcription of Pol?,suggesting that RPA12 and RPA34 could promote the transcription of Pol?.(2)In order to further determine the regulatory effects of the two on Pol? transcription,we constructed the RPA12 overexpression stable cell line and the RPA34 overexpression stable cell line.Through experiments,we found that when these two subunits were overexpressed respectively,the expression levels of some transcription products of Pol? appeared a significant upward-regulation trend.(3)The past studies have proved that Pol? transcription level is closely related to cell proliferation.Through Ed U and CCK8 experiments,it was found that the proliferation rate of cells was inhibited when RPA12 was knocked down,while RPA34 knockdown would not significantly affect the cell proliferation rate,but the overexpression of the two did not affect the cell proliferation rate.(4)We recognized that knockdown of RPA12 affects the morphology of He La,speculating that cell migration might change.Through wound healing experiments and Transwell experiments,it was revealed that the migration rate of He La was accelerated when RPA12 or RPA34 was knocked down,indicating that RPA12 and RPA34 could inhibit cell migration.(5)The changes in the expression levels of Pol?-related transcription factors were analyzed by Western blot.Experimental data revealed that when RPA34 was knocked down,UBF,RPA49,TAFIp48 and TBP existed different degrees of inhibition,and the expression level of some proteins would be up-regulated with the overexpression of RPA34.Knockdown or overexpression of RPA12 in 293 T cells did not affect the expression of UBF,TBP and RPA49.Based on the above experimental data,we found that RPA12 and RPA34 both promote gene transcription directed by Pol?.RPA12 could promote cell proliferation.On the contrary,RPA12 and RPA34 had inhibitory effects on cell migration.Mechanism analysis showed that RPA34 might play a regulatory role in the process of transcription initiation.This study initially explores the biological functions of the two unique subunits of Pol?,and broadens the horizons for further understanding of the role of Pol? subunits in human cells.