Ribosome profiling as a tool to identify putatively coding small open reading frames and peptoswitch mechanisms

S Verbruggen1, V Olexiouk1, D Gawron2, P Van Damme2 and G Menschaert1

  1. BioBix, Lab of Bioinformatics and Computational Genomics, Ghent University, B-9000 Ghent
  2. Department of Medical Protein Research, VIB, B-9000, Ghent

Up to now, small open reading frames (sORFs) were neglected in most prediction tools. However, the genome-wide sequencing of ribosome-protected mRNA fragments (ribosome profiling) enabled a way to detect coding sORFs. Our lab developed a pipeline (called RIBOsORF) for functional sORF identification based on ribosome profiling by combining different tools to assess the coding potential. In that way, different classes of sORFs could be revealed based on their position in the transcriptome. Furthermore, we designed a publically available database ( to store sORF identification results. Based on this database, proteomics datasets from PRIDE were searched for mass spectrometry evidence. A significant share of the sORFs, called the upstream open reading frames (uORF), attracted recent attention as they could regulate the translation of their downstream coding sequence (CDS). The interaction between a canonical CDS and its uORF is called a peptoswitch. In this mechanism, the translation and the ribosomal scanning of the uORF impede the ribosome of translating the CDS downstream. With our in-house PROTEOFORMER pipeline, we determined the ORF based counts in eukaryotic initiation factor 1 knock down cells of the human HCT116 cell line. Out of these counts, a list with the most promising peptoswitch candidates could be constructed and a validation assay with luciferase constructs is now ongoing. Furthermore, we are performing an analogue study in plants, as there is uORF validation available in the new Araport11 annotation.