Identifying Small Open Reading Frames in Prokaryotes with Ribosome Profiling

Abstract

Small proteins encoded by open reading frames (ORFs) shorter than 50 codons (small ORFs [sORFs]) are often overlooked by annotation engines and are difficult to characterize using traditional biochemical techniques. Ribosome profiling has tremendous potential to empirically improve the annotations of prokaryotic genomes. Recent improvements in ribosome profiling methods for bacterial model organisms have revealed many new sORFs in well-characterized genomes. Antibiotics that trap ribosomes just after initiation have played a key role in these developments by allowing the unambiguous identification of the start codons (and, hence, the reading frame) for novel ORFs. Here, we describe these new methods and highlight critical controls and considerations for adapting ribosome profiling to different prokaryotic species.

Keywords: genome annotation; ribosome profiling; sORF; small protein.

FIG 1

(Top) Critical steps in the ribosome profiling protocol and suggestions for how to optimize the protocol for new prokaryotic species. (Bottom) Average ribosome density from yeast Ribo-seq data aligned at start codons and stop codons.

FIG 2

(A) E. coli ribosome density upstream of corA is difficult to reconcile with putative ORFs starting with AUG and GUG start codons. Putative start codons (AUG and GUG) and stop codons (UAA, UAG, and UGA) in each of the three frames are indicated by short and long bars, respectively. The precise start peak in Ribo-seq from retapamulin-treated cells reveals that the density can be attributed to a novel sORF (ysgDI) in frame 1 (dotted arrow) that starts with UUG. The complete sequence of the ysgD gene is shown in orange at the bottom, with the putative SD sequence boxed. The 3′-mapped ribosome footprint data were reported previously (1, 14). (B) Ribosome density on the ndk gene in a negative-control sample and samples treated with the antibiotics tetracycline (Tet), retapamulin (Ret), and apidaecin (Api) (with and without additional puromycin [Pmn] treatment). The 3′-mapped ribosome footprint data were reported previously (14, 15, 50).