Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1

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1 Supplementary Figure 1 Frequency of alternative-cassette-exon engagement with the ribosome is consistent across data from multiple human cell types and from mouse stem cells. Box plots showing AS frequency (scored as the fraction of annotated exons in canonical transcripts that show skipping) for genes with different levels of RNA-Seq or ribosome-profiling read coverage in (a) mouse ES cells, (b) human BJ cells and (c) human HEK293 cells. Simple, complex and microexon (i.e nt) cassette events were analyzed and only genes with detected AS events in RNA-Seq data were included. See Figure 2 and Online Methods for description of boxplots

2 Supplementary Figure 2 Features of cassette exons detected in ribosome profiling and RNA-seq data. a) Bar plot showing the percentage change in detection of AS events in different transcript locations using ribosome profiling and RNA- Seq data. Transcript locations are mapped based on Ensembl GTF annotations 37. b) Bar plot comparing fractions of total alternative 3 and 5 splicing events identified in RNA-Seq data that are also identified as alternative in ribosome profiling (RP) data at increasing expression levels. Events were only analyzed if adjacent constitutive exons were detected in ribosome profiling data. c) Bar plots showing the percentage of alternative exons predicted to be contribute to open reading frames at different expression levels in RNA- Seq data. d) Bar plots showing the percent of exons identified as alternative in both the full dataset and the subsampled datasets. Only reads from genes with crpkm > 250 included. Statistical test used: Fisher s exact test

3 Supplementary Figure 3 Fraction of IR events identified in ribosome profiling data varies depending on transcript location. a) Bar plot showing fraction of total intron retention events identified in RNA-Seq data that are also detected as retained in ribosome profiling (RP) data, for events in locations other than the 5 -UTR and not divisible by 3. Only retention events with supporting evidence in ribosome profiling data are shown. b) Bar plot showing fraction of total intron retention events identified in RNA-Seq data that are also detected as retained in ribosome profiling (RP) data, only for events within 5 -UTR or divisible by 3. Only retention events with supporting evidence in ribosome profiling data are shown. c) Bar plot showing the fraction of genes with 5 -UTR IR events that have annotated upstream open reading frames (uorfs).

4 Supplementary Figure 4

5 Features of periodic AS events. a) Heat map showing the maximum percentage-spliced in (PSI) or percent intron retained (PIR) change between tissues or conditions highlighting events that are tissue-specific and cell-cycle stage-specific. For the analysis, cell-type specific events (Cell Type AS) defined using RNA-Seq 7 that were also detected within ribosome-profiling data and periodic events showed differential cyclic changes as identified by fourier transform analysis. Individual columns were sorted and events matched from right to left to ensure direct comparison of events across cell stages or cell types (see Methods) b) Bar plots comparing the overlap of exons with annotated ATG start sites from Ensembl v71. c) An annotated version of the Enrichment map in Figure 4e. d) Percent of 5 -UTR events that influence upstream open reading frames (uorfs) and Terminal oligopyrimidine tract (TOP) motifs, respectively, for different types (un)regulated events e) Heatmap of PSI/PIR values of Aurora Kinase A (AURKA) and Cell division cycle protein 20 homolog (CDC20) during two rounds of cell cycle. f) Bar plots showing the location of periodic AS events within transcripts based on Ensembl GTF annotation 36, as compared to all detected events from ribosomal profiling. Occurrences in CDS cede to occurrences in UTR. See Figure 2 and Online Methods for description of boxplots. Statistical test used: Fisher s exact test