Benedikt Obermayer | bioRxiv Science

bioRxiv2015

A spectral analysis approach to detect actively translated open reading frames in high-resolution ribosome profiling data

RNA sequencing protocols allow for quantifying gene expression regulation at each individual step, from transcription to protein synthesis. Ribosome Profiling (Ribo-seq) maps the positions of translating ribosomes over the entire transcriptome. Despite its great potential, a rigorous statistical approach to identify translated regions by means of the characteristic three-nucleotide periodicity of Ribo-seq data is not yet available. To fill this gap, we developed RiboTaper, which quantifies the significance of periodic Ribo-seq reads via spectral analysis methods.\n\nWe applied RiboTaper on newly generated, deep Ribo-seq data in HEK293 cells, to derive an extensive map of translation that covers Open Reading Frame (ORF) annotations for more than 11,000 protein-coding genes. We also find distinct ribosomal signatures for several hundred detected upstream ORFs and ORFs in annotated non-coding genes (ncORFs). Mass spectrometry data confirms that RiboTaper achieves excellent coverage of the cellular proteome and validates dozens of novel peptide products. Collectively, RiboTaper (available at https://ohlerlab.mdc-berlin.de/software/) is a powerful method for comprehensive de novo identification of actively used ORFs in the human genome.

Genomics↗

bioRxiv2015

Comprehensive identification and characterization of conserved small ORFs in animals

There is increasing evidence that non-annotated short open reading frames (sORFs) can encode functional micropeptides, but computational identification remains challenging. We expand our published method and predict conserved sORFs in human, mouse, zebrafish, fruit fly and the nematode C. elegans. Isolating specific conservation signatures indicative of purifying selection on encoded amino acid sequence, we identify about 2000 novel sORFs in the untranslated regions of canonical mRNAs or on transcripts annotated as non-coding. Predicted sORFs show stronger conservation signatures than those identified in previous studies and are sometimes conserved over large evolutionary distances. Encoded peptides have little homology to known proteins and are enriched in disordered regions and short interaction motifs. Published ribosome profiling data indicate translation for more than 100 of novel sORFs, and mass spectrometry data gives peptidomic evidence for more than 70 novel candidates. We thus provide a catalog of conserved micropeptides for functional validation in vivo.

Genomics↗