Evolutionäre Konservierung von Proteinfunktionen durch 3'UTR-vermittelte Protein-Protein-Interaktionen
Zusammenfassung der Projektergebnisse
With our protein-centric view of biology, the coding part of mRNAs has been the main focus of attention while the the function of untranslated regions (UTRs) remains poorly characterized. This is despite the fact that alternative 3’UTR expression vastly diversifies the transcriptome and genomics data suggest that the median 3’UTR length is highly correlated to the complexity of organisms. 3’UTRs have been shown to regulate the translational output of mRNAs by modulating mRNA stability and translation rates. Furthermore, 3’UTRs can impact mRNA localization and they can be subject to dynamically regulated epitranscriptomic modifications. More recently, the lab of Dr. Christine Mayr at Memorial Sloan Kettering could show that 3’UTR can also facilitate protein complex formation in human cells and thereby help to organize protein function. In contrast to coding regions, the putative biological information comprised in of 3’UTRs is more difficult to decode as it is composed of both structural and sequence information which act together in a combinatorial fashion. Meanwhile, tools to systematically investigate 3’UTR functions are less developed and this further contributes to our our general lack of understanding of their role. The advent of the CRISPR-Cas9 technology has opened the door for new approaches to overcome these problems. I could show that targeted recruitment of CRISPR-Cas9 can be used to generate cell lines that can be made to either express specific 3’UTR isoforms or have a deletion of the entire 3’UTR. In the future, these approaches will enable a more detailed analysis of 3’UTR function across different organisms. I used this approach to generate cells that lack the 3’UTR of the tumor suppressor gene p53 in a well established human carcinoma cell line. Unexpectedly, these cells expressed normal amounts of p53 protein in steady state as well as after exposure to DNA-damaging agents. These data challenge earlier assumptions that the 3’UTR of p53 is important for protein expression regulation. However, despite normal p53 protein levels I found that p53 target genes displayed elevated expression in cells lacking the 3’UTR both in steady state and after moderate DNA damage. I also found that p53 protein that is expressed in cells lacking the 3’UTR, exhibits a higher level of baseline binding to target gene promoters. Phenotypically, TP53dUTR cells display increased sensitivity for growth arrest and senescence when stimulated with DNA damaging drugs at low or moderate doses. Currently, I am working towards identifying the proteins that are responsible for repressing p53 activity in steady state through a 3’UTR- dependent mechanism. I hope that this research will help to better understand the biology of this important tumor suppressor and could open up new avenues for treatments that interfere with RNA binding.
