Abstract
UPF1 eliminates aberrant mRNAs harboring premature termination codons, and regulates the steady-state levels of normal physiological mRNAs. Although genome-wide studies of UPF1 targets performed, previous studies did not distinguish indirect UPF1 targets because they could not determine UPF1-dependent altered RNA stabilities. Here, we measured the decay rates of the whole transcriptome in UPF1-depleted HeLa cells using BRIC-seq, an inhibitor-free method for directly measuring RNA stability. We determined the half-lives and expression levels of 9,229 transcripts. An amount of 785 transcripts were stabilized in UPF1-depleted cells. Among these, the expression levels of 76 transcripts were increased, but those of the other 709 transcripts were not altered. RNA immunoprecipitation showed UPF1 bound to the stabilized transcripts, suggesting that UPF1 directly degrades the 709 transcripts. Many UPF1 targets in this study were newly identified. This study clearly demonstrates that direct determination of RNA stability is a powerful approach for identifying targets of RNA degradation factors.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Dr. Akio Yamashita (Yokohama City University) for providing the UPF1 expression vector. We thank MBL Co., Ltd for donating the anti-BrdU antibody. This work financially supported by the Takeda Science Foundation, the Naito Foundation, Grants-in-Aid for Scientific Research, Research Fellowship of the Japan Society for the Promotion of Science, the Funding Program for World-Leading Innovative R&D on Science and Technology of the Japan Society for the Promotion of Science, and Grant-in-Aid for Scientific Research on Innovative Areas 'Functional machinery for non-coding RNAs' and 'Genome Science' from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Supplemental Materials
Supplemental materials may be found here: https://www.landesbioscience.com/journals/rnabiology/article/22360/