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Fig. s5 A. Histogram showing the frequency of all APPRIS 3′UI-containing transcripts in human GRCh38 as a measure of the distance of the 3′UI from the stop codon. Data are grouped in 5 nt bins from 1–500 nts. Proximal 3′UI-containing gene bins are indicated in blue; distal 3′UI-containing gene bins are indicated in gray. Red dotted line indicates distance from stop codon to farthest 3′UI = 50 nts. Histogram of transcripts from Ensembl annotation is shown in C. B. Histogram as in A of mouse proximal and distal 3′UI-containing transcripts in mouse GRCm38. Histogram of transcripts from Ensembl annotation is shown in D. C. Histogram as in A of proximal and distal 3′UI-containing human transcripts from Ensembl annotation of GRCh38. D. Histogram as in A of proximal and distal 3′UI-containing mouse transcripts from Ensembl annotation of GRCm38. E. PANTHER14.0 [86] gene ontology (GO) term enrichment analysis of human APPRIS proximal 3′UI-containing genes (shades of blue) and all human APPRIS 3′UI-containing genes (shades of gray). All significant terms (Benjamini-Hochberg corrected p-value < 0.05) are shown for each set. F. GO term enrichment analysis as in E of mouse APPRIS 3′UI-containing genes. G. A scatter plot showing gene-level fold changes for all Ensembl-annotated transcripts with proximal 3′UI (dark blue: FC > 1.5 and light blue: FC < 1.5) and distal 3′UI (black: FC > 1.5 and gray: FC < 1.5) in UPF1 knockdown human embryonic kidney cells (HEK293) compared to control cells using previously published data [57]. Genes encircled in red also contain a uORF as determined from a previously published dataset (see Methods). H. A scatter plot showing gene-level fold changes for all APPRIS-annotated transcripts with proximal 3′UI (dark blue: FC > 1.5 and light blue: FC < 1.5) and distal 3′UI (black: FC > 1.5 and gray: FC < 1.5) in UPF1 knockdown human embryonic stem cells (hESCs) compared to control cells using previously published data [62]. Genes encircled on red also contain a uORF as determined from a previously published dataset (see Materials and Methods). I. Scatter plot as in E showing gene-level fold changes of APPRIS-annotated mouse 3′UI-containing transcripts in Smg6-/- knockout mouse embryonic stem cells (mESCs) compared to wild-type cells [63]. J. Western blots showing representative protein knockdown in one replicate for Fig 7E.