基因组与蛋白学技术RNA组学.ppt

* So we have showed fip1 kd caused 3’ utr lengthening ,next we asked how 3’ utr lengthening affects the target gene expression, to this purpose.. We selected a panel of Fip1 target genes and cloned their short/constitutive 3’ UTRs or their long/extended 3’ UTRs from different APA isoforms downstream of a reporter genes. Then we used these constructs in luciferase assays and found that, in 7 out of 8 genes, the longer 3’ UTR lead to lower expression than the shorter 3’ UTRs. To check the effect of APA changes on the endogenous gene expression, we have checked the protein levels of several genes that showed 3’ UTR lengthening when Fip1 is depleted. * * To determine the biological significance of Fip1 regulated APA, we characterized the functions of Fip1 APA targets. Interestingly, there is an significant enrichment of genes that function in cell cycle, proliferation and development. * * Therefore, Fip1 depletion induces 3’ UTR lengthenging which in turn leads to lower expression of many that may have important functions in controlling proliferation and differentiation in stem cells. To directly test if Fip1 APA target genes are important for ES cells, we knocked down some of these genes. Interestingly, similar to Fip1 depletion, knockdown of Fip1 APA target genes also resulted in ES cell differentiation. These results suggest that Fip1 promotes ESC self-renewal by regulating the APA and expression of critical self-renewal factors. * * Many self-renewal factors are also important for reprogramming of differentiated cell to iPS cells. So next we wanted to determine if Fip1 plays a role in reprogramming. So we took mouse embryonic fibroblast cells and introduced control shRNAs for Fip1-specific shRNAs. Interestingly, Fip1 knockdown did not seem to have a significantly effect on cell growth in MEF. Strikingly, however, Fip1 depletion caused a near 10-fold decrease in reprogramming efficiency as you can see here with the AP staining and the quantification. This result dem