Fig 2: MDM4 and RPL22L1 are splicing targets of RPL22(A) Total RPL22L1 mRNA expression across experiments. Bars represent the mean of three replicates (**p < 0.01).(B) Common differentially expressed genes across RPL22 knockout experiments. The columns and numbers indicate the total counts of differentially expressed genes in each combination of intersections between the different experiments.(C) Inclusion proportions of RPL22L1 exon 3A across experiments. RPL22 knockdown reduces RPL22L1 exon 3A inclusion. Bars represent the mean of three replicates (**p < 0.01). PSI, percent spliced in.(D) Changes in splicing modes across experiments as indicated by significant differentially spliced variants. The numbers in parentheses indicate the total counts of differential splicing events in each experiment. A3SS, alternative 3’ splicing site; A5SS, alternative 5’ splicing site; MXE, mutually exclusive exons; RI, retained intron; SE, skipped exon.(E) Common splicing changes across RPL22 knockout experiments. The columns and numbers indicate the total counts of differentially expressed exons in each combination of intersections between the different experiments.(F) Inclusion levels of MDM4 exon 6 across experiments. Bars represent the mean of three replicates (**p < 0.01).

Fig 3: Characterization of RPL22 binding preference with CLIP-seq(A) Binding of RPL22 to the 3’ UTR and exon 6 of MDM4 as determined by peak calling on CLIP-seq measurements from ZR-75–1 cells. p = 0.05.(B) Left: peak frequencies relative to the transcription termination site (TTS). Right: breakdown of peak-containing regions.(C) Gene set enrichment analysis of RPL22-bound peaks.(D) Overlaps between genes containing RPL22-bound peaks and differentially spliced genes across RPL22 modulation experiments. Total number of genes and hypergeometric test for each cell line: LNCaP: 15,602 genes, p = 1.536 × 10−4; NCI-H2110: 15,226 genes, p = 6.682 × 10−9; and ZR75–1: 15,690 genes, p = 8.901 × 10−6.(E) Schematic model of loss of RPL22 and recurrent RPL22 frameshift mutations in MSI-H tumors that results in a splicing-mediated switch to the functional isoforms of MDM4 and RPL22L1. The functional isoform of MDM4 inhibits p53 activity and increased levels of RPL22L1 compensate for RPL22 reduction in the ribosome and perform additional extraribosomal functions.