UAP56 associates with DRM2 and is localized to chromatin in Arabidopsis

Repeated sequence expression and transposable element mobilization are tightly controlled by multilayer processes, which include DNA 5′‐cytosine methylation. The RNA‐directed DNA methylation (RdDM) pathway, which uses siRNAs to guide sequence‐specific directed DNA methylation, emerged specifically in plants. RdDM ensures DNA methylation maintenance on asymmetric CHH sites and specifically initiates de novo methylation in all cytosine sequence contexts through the action of DRM DNA methyltransferases, of which DRM2 is the most prominent. The RdDM pathway has been well described, but how DRM2 is recruited onto DNA targets and associates with other RdDM factors remains unknown. To address these questions, we developed biochemical approaches to allow the identification of factors that may escape genetic screens, such as proteins encoded by multigenic families. Through both conventional and affinity purification of DRM2, we identified DEAD box RNA helicases U2AF56 Associated Protein 56 (UAP56a/b), which are widespread among eukaryotes, as new DRM2 partners. We have shown that, similar to DRM2 and other RdDM actors, UAP56 has chromatin‐associated protein properties. We confirmed this association both in vitro and in vivo in reproductive tissues. In addition, our experiments also suggest that UAP56 may exhibit differential distribution in cells depending on plant organ. While originally identified for its role in splicing, our study suggests that UAP56 may also have other roles, and our findings allow us to initiate discussion about its potential role in the RdDM pathway. Two biochemical purification strategies were used to identify the DEAD box RNA helicases UAP56a/b as new DRM2 partners; UAP56a/b are known to affect splicing and export of polymerase II (Pol II)‐dependent transcripts in eukaryotes. In this context, UAP56 proteins are proposed to contribute to the stabilization of DRM2 close to Pol II or Pol V‐targeted silenced loci in Arabidopsis.