Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing Other
Summary
Topoisomerase 3β (TOP3B) and TDRD3 form a dual-activity topoisomerase complex that interacts with FMRP and can change the topology of both DNA and RNA. Here, we investigated the post-transcriptional influence of TOP3B and associated proteins on mRNA translation and turnover. First, we discovered that in human HCT116 colon cancer cells, knock-out (KO) of TOP3B had similar effects on mRNA turnover and translation as did TDRD3-KO, while FMRP-KO resulted in rather distinct effects, indicating that TOP3B had stronger coordination with TDRD3 than FMRP in mRNA regulation. Second, we identified TOP3B-bound mRNAs in HCT116 cells; we found that while TOP3B did not directly influence the stability or translation of most TOP3B target mRNAs, it stabilized a subset of target mRNAs but had a more complex effect on translation--enhancing for some mRNAs whereas reducing for others. Interestingly, a point mutation that specifically disrupted TOP3B catalytic activity only partially recapitulated the effects of TOP3B-KO on mRNA stability and translation, suggesting that the impact of TOP3B on target mRNAs is only in part linked to its ability to change topology of mRNAs. Collectively, our data suggest that TOP3B-TDRD3 can regulate mRNA translation and turnover by mechanisms that are dependent and independent of topoisomerase activity.
Overall design
To identify the functions of the TOP3B-TDRD3 complex on post-transcriptional regulation in HCT116 cells, total 62 samples were uploaded, including PRO-seq, RNA-seq, Ribo-seq and eCLIP-seq samples from WT and KO/KI cells. Most of them contain two or three replications. For TOP3B eCLIP-seq, TOP3B-KO cells were used as negative controls separately. For PRO-seq, RNA-seq and Ribo-seq, wild type cells were used as controls.
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