Figure 4.

Ribo-seq combined with MS expand the H. volcanii small proteome. (A). Overlap between the novel small proteins detected by Ribo-seq (blue) and MS (grey). (B). Length distribution (in codons) of the novel small proteins identified by Ribo-seq. (C). and (D). In vivo validation of translation for five novel small proteins identified either by both Ribo-seq and MS (sORF8, 56 aa; sORF10, 40 aa, and sORF13, 45 aa; top panel) (C) or only by Ribo-seq (sORF46, 42 aa and sORF47, 23 aa; bottom panel) (D). Small ORFs were C-terminally fused to a 3xFLAG tag and expressed from a plasmid in H. volcanii. Strains were grown to exponential phase in selective media and protein extracts were analyzed by western blotting. Proteins were detected with an anti-FLAG antibody. A nontranslated sORF served as negative control (Figure S5D, Supporting Information). M: molecular weight marker. Top: genome browser screenshots of read coverage from Ribo-seq(blue track)/RNA-seq(black track) libraries. Genomic positions are indicated below with a schematic representation of the genomic region (novel sORFs in black). Bent arrows indicate the transcription start sites (TSS) based on Babski et al. (2016).