Fig 2: UBR4 Regulates a Set of Muscle Proteins that Physically Interact with and Are Ubiquitinated by UBR4(A) TMT proteomics and RNA-sequencing from TA muscles electroporated with two different UBR4 siRNAs and examined 7 days after electroporation, compared to contralateral muscles electroporated with a non-targeting (NT) control siRNA. log2 fold changes (averaged from three biological replicates) are shown. Increasing circle size represents higher statistical significance. Red highlights putative UBR4 targets, i.e., proteins significantly regulated (> 50% change with p < 0.05) by both siRNAs in proteomics experiments. Note that only few of these are also transcriptionally regulated (n = 3 separate muscle biological replicates).(B) Putative UBR4 target proteins found to be significantly regulated in TA muscles are overall consistently regulated in C2C12 cells transfected with UBR4 siRNAs (n = 3 separate biological replicates).(C) Western blots and protein quantification confirm upregulation of the putative UBR4 target proteins UBE2B, HAT1, RBBP4/7, PCNA, and STUB1 in different model systems with loss of UBR4, i.e., C2C12 treated with UBR4 siRNAs, TA muscles electroporated with UBR4 siRNAs, and TA and soleus muscles from UBR4 mKO mice and controls.(D) Diagram of FLAG-tagged UBR4 N- and C-terminal domains used for co-immunoprecipitation experiments following transfection into HEK293T cells. FLAG affinity purification of UBR4N and UBR4C retrieved associated proteins, which were identified by mass spectrometry, and are indicated in the heatmap as significantly enriched (red; p < 0.05) or not (blue). See Tables S4 for the corresponding primary data.(E) Co-immunoprecipitation of FLAG-tagged UBR4 target proteins expressed in HEK293T cells treated with either NT control or UBR4 siRNAs and 50-μM MG132 for 4 h. Full western blots with UBE2B-FLAG are shown as an example. Both input and immunoprecipitation (IP) samples are probed with anti-UBR4 antibodies to detect pull-down of endogenous UBR4, and with anti-ubiquitin antibodies to determine whether putative UBR4 target proteins are differentially ubiquitinated in response to NT and UBR4 siRNAs. Similar to UBE2B, IP of the putative UBR4 targets HAT1, RBBP4, and RBBP7 retrieves endogenous UBR4, and UBR4 knockdown reduces the ubiquitination of these proteins, compared to control samples. Ubiquitin (Ub)/FLAG ratios indicate changes in ubiquitination relative to total FLAG protein levels with UBR4 knockdown.(F) Immunoprecipitation of FLAG-tagged UBR4 target proteins in HEK293T cells that express either full-length human V5-tagged UBR4 or carry a control empty vector. UBE2B and HAT1 co-immunoprecipitate with UBR4-V5. Ub/FLAG ratios indicate changes in ubiquitination relative to total FLAG protein levels with UBR4-V5 expression.

Fig 3: RNAi Screening for Ub Ligases that Regulate Myofiber Size Identifies a Key Role for UBR4 in Myofiber Hypertrophy in Drosophila and MiceThe steady-state control of myofiber size depends on the modulation of the levels of UBR4 protein targets. The E2 Ub conjugating enzyme UBE2B acts in concert with the E3 Ub ligase UBR4 to mediate ubiquitination and proteasomal degradation of UBR4 substrates. UBR4 loss induces myofiber hypertrophy through increased levels of multiple UBR4 protein targets (such as the HAT1/RBBP4/RBBP7 histone-binding complex) and diverse mechanisms, including the HAT1-dependent expression of genes that sustain hypertrophy, such as sarcolipin (Sln).

Fig 4: AID 2.1 enables minimal basal degradation, efficient target depletion, and faster dynamic recovery.a–c The western blots show basal and induced depletion of degron-tagged endogenous DNMT3B, U2AF2, and RBBP4 proteins in iPSCs expressing AID 2.0 (OsTIR1F74G) and AID 2.1 (OsTIR1F74G+S210A) after being treated with 1 μM 5-Phenyl-indole-3-acetic acid (5-Ph-IAA) for the indicated times. The bar plots show quantification of basal protein levels in both systems from three independent biological replicates. d The line plot shows Incucyte live cell imaging data indicating relative apoptosis rates in degron-tagged-RBBP4 iPSCs expressing OsTIR1F74G or OsTIR1F74G+S210A after DMSO control and 1 μM 5-Ph-IAA treatment. e The line plots show relative cell proliferation rates in degron-tagged-RBBP4 iPSCs expressing OsTIR1F74G or OsTIR1F74G+S210A and the wild-type parental cells without auxin treatment. f Western blots show the basal RBBP4 protein levels in degron-tagged-RBBP4 iPSCs expressing OsTIR1F74G or OsTIR1F74G+S210A. g The volcano plots show differential gene expression levels in degron-tagged RBBP4mAID/mAID iPSCs expressing AID 2.0 (left panel) or AID 2.1 (right panel) relative to the parental RBBP4WT/WT iPSCs. h Colony formation assay (quantified in bar plot) shows the remaining surviving iPSCs after ligand washout (after 6 h of 1 μM 5-Ph-IAA treatment) in degron-tagged-RBBP4 iPSCs expressing AID 2.0 or AID 2.1. i The Incucyte live cell imaging results show relative apoptosis rates in degron-tagged-RBBP4 iPSCs expressing AID 2.0 or AID 2.1 after auxin washout (after 3 h of 5-Ph-IAA treatment). Data and error bars in a–e and g–i indicate the means ± SD (n = 3 biological replicates in (a, b, d, e, g–i), n = 4 biological replicates in (c)). A two-sided unpaired t-test has been used for statistical analysis. Source data are provided as a Source Data file.