The midnolin-proteasome pathway catches proteins for ubiquitination-independent degradation

Cells use ubiquitin to mark proteins for proteasomal degradation. While the proteasome also eliminates proteins that are not modified by ubiquitin, how this occurs mechanistically is unclear. We show here that midnolin promotes the destruction of many nuclear proteins including transcription factors encoded by the immediate-early-genes. Diverse environmental cues induce midnolin and its overexpression is sufficient to cause the degradation of its targets by a mechanism, which, remarkably, does not require ubiquitination. Instead, midnolin associates with the proteasome via an alpha-helix, employs its Catch-domain to bind a region within substrates that adopts a beta-strand conformation, and uses a ubiquitin-like-domain to promote substrate destruction. Thus, midnolin contains three regions that function in concert to target a large set of nuclear proteins to the proteasome for degradation. , DataS1: EGR1 and FosB genome-wide CRISPR-Cas9 screens
Genome-wide CRISPR-Cas9 screens were performed to uncover regulators of EGR1 and FosB protein stability. Specifically, the plasmid library was packaged into lentivirus by transfecting HEK-293T cells using PolyJet as described earlier, and the lentivirus was titered to obtain a multiplicity of infection around 0.3. HEK-293T cells were generated to express the GPS 3.0 FosB or GPS 3.2 EGR1 reporters by selecting using hygromycin (200 µg/mL). These cells were then transduced with the titered CRISPR-Cas9 genome-wide Root library lentivirus at an MOI ~0.3 to maintain a 500x representation throughout. Cells were selected 48 hours post-transduction for 7 days using puromycin (2 µg/mL) to remove uninfected cells. On the ninth day of puromycin selection, the 95th percentile most stable cell population was collected based on the GFP/DsRed ratio by FACS using a MoFlo Astrios instrument (Beckman Coulter). Additionally, the unsorted input cells we..., PDB files for AlphaFold multimer predictions can be opened with softwares such as PyMOL and ChimeraX.Â