1. Supplemental Figure S1
Supplemental Figure S1. (A) Schematic shows the three KREN1, KREN2, and KREN3 ~20S editosomes, and their protein interactions identified by yeast two-hybrid and co-expression studies (dashed lines) (Schnaufer et al. 2003; Schnaufer et al. 2010; Mehta et al. 2015). (B-D) Networks show detailed editosome architecture revealed by cross-linking and mass spectrometry (CXMS) (McDermott et al. 2016). Network edge widths are proportional to the number of interlinks observed between two proteins. All previously described interactions between editosome proteins were found in our cross-linking data. (B) All interlinks, (C) KREPB4 first neighbors only, and (D) KREPB5 first neighbors only. (E) Interprotein cross-linking maps of KREPB4 first neighbors that contain RNase III domains which do (KREN1, KREN2, KREN3) or do not (KREPB5, KREPB6, KREPB7, KREPB10) have conserved catalytic residues. Black dots distributed across proteins indicate the positions of lysine residues available for cross-linking, and domains are highlighted as indicated based on previous studies or bioinformatics predictions. 

2. Supplemental Figure S2
Supplemental Figure S2. (A) Western analysis with anti-V5 tag monoclonal antibody showing expression of V5-tagged mutant KREPB4 proteins from the β-tubulin locus in BF and PF CN cells (equivalent of 4 x 106 and 2 x 106 cells/lane respectively) in the presence and absence of tet. (B) RT-qPCR analysis of BF and PF CN cells that constitutively express WT, or a PUF-triple (T280A/H281A/E284A) mutant version of KREPB4, and in which the tet-regulatable WT KREPB4 allele was expressed (E) or repressed (R) for 2 and 4 days respectively. The abundances of transcripts from KREPB4 alleles (B4 reg=tet-regulatable WT allele; B4 EE=exclusively expressed V5-tagged WT or mutant allele; B4 ORF=B4 open reading frame), and never-edited (COI and ND4), pre-edited, and edited mitochondrial mRNAs in repressed cells were calculated relative to cells expressing tet-regulatable WT KREPB4. For each target amplicon, relative abundance was determined using TERT as an internal control. Heat map shows the log10-transformed relative abundances of RNAs from BF cells (left) and PF cells (right), as indicated by the scale bar (bottom). Data represent mean of two experiments. (C) Data from (B), presented as means ± SEM, plotted on a log10 scale.

3. Supplemental Figure S3
Supplemental Figure S3. Analysis of the growth of KREPB4 CN cells containing WT or putative PTM mutant versions of KREPB4. Cumulative growth of BF and PF CN cells constitutively expressing V5-tagged WT or PTM mutant versions of KREPB4, and in which the tet-regulatable WT KREPB4 allele was expressed (E) or repressed (R). Exclusive expression of WT and all PTM mutant proteins (each indicated by R) permitted normal growth in BF and PF.