Protein ubiquitination, a major post-translational modification in eukaryotes, 
requires an adequate pool of free ubiquitin. Cells maintain this pool by two 
pathways, both involving deubiquitinases (DUBs): recycling of ubiquitin from 
ubiquitin conjugates and processing of ubiquitin precursors synthesized de novo. 
Although many advances have been made in recent years regarding ubiquitin 
recycling, our knowledge on ubiquitin precursor processing is still limited, and 
questions such as when are these precursors processed and which DUBs are 
involved remain largely unanswered. Here we provide data suggesting that two of 
the four mammalian ubiquitin precursors, UBA52 and UBA80, are processed mostly 
post-translationally whereas the other two, UBB and UBC, probably undergo a 
combination of co- and post-translational processing. Using an unbiased 
biochemical approach we found that UCHL3, USP9X, USP7, USP5 and 
Otulin/Gumby/FAM105b are by far the most active DUBs acting on these precursors. 
The identification of these DUBs together with their properties suggests that 
each ubiquitin precursor can be processed in at least two different manners, 
explaining the robustness of the ubiquitin de novo synthesis pathway.