ABSTRACT
Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.
Subject(s)
Brain/abnormalities , Cytosine/analogs & derivatives , Drosophila melanogaster/growth & development , RNA, Messenger/metabolism , 5-Methylcytosine/analogs & derivatives , Animals , Brain/metabolism , Cell Line , Cytosine/metabolism , Dioxygenases/genetics , Dioxygenases/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Methylation , RNA, Messenger/genetics , TranscriptomeABSTRACT
The TIS11/tristetraprolin (TTP) CCCH tandem zinc finger proteins are major effectors in the destabilization of mRNAs bearing AU-rich elements (ARE) in their 3' untranslated regions. In this report, we demonstrate that the Drosophila melanogaster dTIS11 protein is short-lived due to its rapid ubiquitin-independent degradation by the proteasome. Our data indicate that this mechanism is tightly associated with the intrinsically unstructured, disordered N- and C-terminal domains of the protein. Furthermore, we show that TTP, the mammalian TIS11/TTP protein prototype, shares the same three-dimensional characteristics and is degraded by the same proteolytic pathway as dTIS11, thereby indicating that this mechanism has been conserved across evolution. Finally, we observed a phosphorylation-dependent inhibition of dTIS11 and TTP degradation by the proteasome in vitro, raising the possibility that such modifications directly affect proteasomal recognition for these proteins. As a group, RNA-binding proteins (RNA-BPs) have been described as enriched in intrinsically disordered regions, thus raising the possibility that the mechanism that we uncovered for TIS11/TTP turnover is widespread among other RNA-BPs.