N-Terminally arginylated ubiquitin is attached to histone H2A by RING1B E3 ligase in human cells.

Ubiquitin (Ub) is highly conserved in all eukaryotic organisms and begins at the N-terminus with Met and Gln. Our recent research demonstrates that N-terminally (Nt-) arginylated Ub can be produced in the yeast Saccharomyces cerevisiae. However, the existence of Nt-arginylated Ub in multicellular organisms remains unknown. Here we explore the mechanism for creating Nt-arginylated Ub using human embryonic kidney HEK293 cells that express various Nt-modified Ubs. We found that Gln-starting Q-Ub was converted into Glu-starting E-Ub by NTAQ1 Nt-deamidase and subsequently Nt-arginylated by ATE1 arginyltransferase in HEK293 cells. We also found that the resulting Arg-Glu-starting RE-Ub was mainly deposited on the Lys119 residue of histone H2A. Furthermore, RING1B E3 Ub ligase mediated the attachment of RE-Ub to H2A. These findings reveal a previously unknown type of histone ubiquitylation which greatly increases the combinatorial complexity of histone and ubiquitin codes.