The aim of our proposal is to elucidate the mechanisms that link transcription of specific RNAs in the nucleus to their translation (RNA processing). We have identified and characterized a novel and highly conserved gene, Zfrp8/PDCD2, and shown that it is essential in stem cells in flies, mouse, and human, and that it is also required for growth of cancer cells. We discovered Zfrp8/PDCD2 is required for the nuclear export of select mRNAs and TE transcripts. Also it interacts with the small ribosomal subunit and forms a complex with mRNA binding proteins. Our data suggest that Zfrp8/PDCD2 controls subcellular localization of select RNAs and the association of mRNARNPs with ribosomes. We also have identified Tet/TET1 as a new Zfrp8/PDCD2 interacting protein. In vertebrates, TET proteins function in DNA demethylation converting 5methylcytosine (5mC) into 5hydroxymethylcytosine (5hmC), modifications that are not detected in Drosophila DNA. A recent study shows that vertebrate Tet proteins can also convert 5mrC to 5hmrC in RNA. Inspired by this discovery, we have shown that 5hmrC also exists in flies and depends on Tet activity. We hypothesize that Tet modifies specific transcripts and regulates the recruitment of Zfrp8 to these RNAs, so controlling their processing and translation. We propose to test this hypothesis by a combination of molecular/biochemical and genetic experiments. We will identify 5hmrCmodified transcripts transcriptomewide and study their integrity, levels, and localization in wild type and mutant tissues. We will map the Tet binding sites on DNA and compare their location to 5hmrCmodified transcripts. Finally, we will test how Tet and Zfrp8 affect ribosomal occupancy of mRNAs and establish how Tet and 5hmrC affect their translation. In addition to investigating the mechanism by which Tet and 5hmrC regulate RNA metabolism and how Zfrp8 affects the process, we will determine the importance of both genes in development and stem cell differentiation by studying the mutant phenotypes of new Tet alleles and Tet and Zfrp8 KD tissues. Because of the conservation of both Tet and Zfrp8/PDCD2 our results are likely to shed light on the same process in mouse and human.
|Effective start/end date||9/1/16 → 5/31/20|
- National Institutes of Health (NIH)