Lysine-independent ubiquitination and degradation of REV-ERBα involves a bi-functional degradation control sequence at its N-terminus.

REV-ERBα and REV-ERBβ are important components of the mammalian circadian clock and play a crucial role in linking the circadian system to overt daily rhythms in physiology and behavior. Expression of these parologs is driven by the circadian clockwork, and in most tissues, the abundance of REV-ERBα proteins robustly cycles such that they are detected only within a tight interval of 4-6 hours each day, suggesting control of both their synthesis and degradation are tightly controlled. Indeed, several different ubiquitin ligases have been shown to mediate REV-ERBα degradation, but how they interact with REV-ERBα and which lysine residues they ubiquitinate to drive its degradation are unknown. Here, we used a mutagenesis approach to functionally identify both binding and ubiquitination sites within REV-ERBα required for its regulation by the ubiquitin ligases Spsb4 and Siah2. Surprisingly, we found that REV-ERBα mutants with all 20 lysines changed to arginine (K20R) can be efficiently ubiquitinated and degraded in the absence or presence of these E3 ligases, consistent with N-terminal ubiquitination. To explore this, we examined if small deletions at the N-terminus of REV-ERBα will alter its degradation. Interestingly, deletion of amino acid (AA) residues 2 to 9 (delAA2-9) clearly resulted in a less stable REV-ERBα. We found that it was the length (i.e. 8 AA), and not the specific sequence, that confers stability in this region.. Simultaneously, we also mapped the interaction site of the E3 ligase Spsb4 to this same regions, specifically requiring AA4-9 of REV-ERBα. Thus, the first 9 AA of REV-ERBα has two opposing roles in regulating REV-ERBα turnover. Additionally, deleting eight addition additiona AAs (delAA2-17) from REV-ERBα almost prevents its degration. Combined, these results suggest that complex interactions within the first 25AAs that potentially act as a REV-ERBα 'switch' that allows a protected/stabilized conformation to accumulate at one time of day, but then rapidly shifts to a destabilized form, to enhance its removal at the end of the daily cycle.