Phosphorylation event changes the RNA binding mode of EZH2 disordered segment.

Polycomb repressive complex 2 (PRC2), which exhibits an important gene silencing role in development and differentiation, has been shown to interact with several long non-coding RNAs (lncRNAs) that influence its function and determine its localization. One of the most prominent and researched lncRNA partners of PRC2 is HOTAIR, which is shown to direct the proper localization of the complex within the chromatin. While many important details of the recognition of RNAs by different complex subunits have been revealed, the exact molecular mechanisms remain elusive. EZH2, the methyltransferase subunit of PRC2, is one of the proteins that are responsible for RNA binding, containing multiple RNA binding surfaces. One of the binding interfaces lies in a disordered loop region of EZH2, where a phosphorylation event is known to increase RNA binding in cells. To reveal the molecular details of the effect of phosphorylation of the disordered loop of EZH2, we expressed the region and tested its interaction with several RNA constructs, including different segments of HOTAIR. We found that the EZH2 loop exhibits varying affinities towards different RNA species, with a limited sequence specificity. Phosphorylation did not have a significant effect on binding strengths, but it altered the structural background of the interaction. While the protein itself remained disordered in the bound form, the phosphomimetic mutant version of the EZH2 loop was capable of opening (unfolding) the double-stranded RNA regions upon interaction. Our findings offer an explanation of the molecular mechanism of the RNA recognition by a disordered segment in EZH2 and clarify the assumed regulatory role of the phosphorylation event.