| GW182 and argonaute 2(AGO2)are core proteins of the RNA interference complex.The TNRC6 paralogs associate with argonaute(AGO)protein,the core RNAi factor,and bridge its interactions with other proteins.GW182,known as TNRC6 A,has the other two paralogs,TNRC6 B and TNRC6 C.Researchers have been worked on the biological effects of AGO2,however,no further research or fundamental understanding about TNRC6 A,even the TNRC6 protein family have been made in cells.With the robust and rapidly developing research methods,we can use siRNAs to knock down gene expression level,or we can knock out the genes to study their contributions in the cell.To investigate the TNRC6 family,we use the siRNAs to knock down TNRC6 gene expression level in different cell lines.Here we test the necessity for GW182 proteins(paralogs TNRC6 A,TNRC6B,and TNRC6C)for several mechanisms of small duplex RNA-mediated control of gene expression,including translational silencing by miRNAs,translational silencing by siRNAs,transcriptional silencing,transcriptional activation,and splicing.Only AGO2 is necessary for each of these processes.GW182 is required for some actions.Also,the gene knocks out cell lines were obtained through the CRISPR/Cas9 system,mass spectrometry used for determining the protein copy numbers per cell to validate the knockout.We test the Following are the main results in this study:(1)We use siRNAs successfully knocking down the TNRC6 paralogs' expression.And test the distribution in the cells.We found that GW182 is required for transcriptional activation and the activity of miRNAs but is dispensable for the regulation of splicing,transcriptional silencing,and the action of siRNAs.AGO2,by contrast,is necessary for each of these processes.(2)We obtain the TNRC6 A single knockout,TNRC6 B single knockout and TNRC6 AB double-knockout cell lines via the CRISPR/Cas9 system investigate how the TNRC6 paralogs contribute to RNAi.We tried lots of times and failed to obtain the TNRC6 ABC triple knockout cells,also found that the cells growing slower significant after knockout TNRC6 AB together.We are pretty sure that the TNRC6 paralogs affect cell growth.Mass spectrometry and qPCR were used here to test the gene expression level on protein and mRNA level.We also observed the TNRC6 A and AGO2 protein distribution in cytoplasm and nucleus via immunostaining.(3)Taking the advantage of knockout cell lines,we tested that TNRC6 A can affect the Dicer localization in cytoplasm versus the nucleus,but none of the three TNRC6 paralogs was necessary for nuclear localization of AGO2.We found that TNRC6 proteins are not required for gene silencing when duplex RNAs are fully complementary.TNRC6 expression was necessary for regulation by a miRNA.TNRC6 A,but not TNRC6 B,expression was necessary for transcriptional activation by a duplex RNA targeting a gene promoter.By contrast,AGO2 is required for all three gene expression pathways.(4)We also did the RNA-seq on all the knockout cell lines.We found that in the TNRC6 A knockout cells,1447 genes' expression upregulated significantly and 1190 genes' downregulated,the upregulated genes focus on developmental growth,surface receptor and et.al.In the TNRC6 B knockout cells,only 270 genes' expression upregulated and 156 genes' downregulated.In the TNRC6 AB knockout cells,2750 genes' expression upregulated and 2710 genes' downregulated,the upregulated genes base on developmental growth,surface receptor,cell-substrate adhesion and et.al,the down regulated genes focus on ribonucleoprotein complex-relative,ribosome RNA and et.al.We can see that TNRC6 A is more important than TNRC6 B gene,but they also supplement each other contribute to the cells.All in all,TNRC6 plays a very important role in cells especially a decisive role in miRNA-mediated gene silencing.It is also involved in transcriptional activation of some genes.Moreover,the results of mass spectrometry and RNA-seq provide the basis for the identification of the role of TNRC6 gene in the overall level of mammalian cells. |
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