title: Extensive identification and analysis of conserved small ORFs in animals.
abstract: BACKGROUND
  There is increasing evidence that transcripts or transcript regions annotated as non-coding can harbor functional short open reading frames (sORFs). Loss-of-function experiments have identified essential developmental or physiological roles for a few of the encoded peptides (micropeptides), but genome-wide experimental or computational identification of functional sORFs remains challenging.
  
  
  RESULTS
  Here, we expand our previously developed method and present results of an integrated computational pipeline for the identification of conserved sORFs in human, mouse, zebrafish, fruit fly, and the nematode C. elegans. Isolating specific conservation signatures indicative of purifying selection on amino acid (rather than nucleotide) sequence, we identify about 2,000 novel small ORFs located 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. The encoded peptides have little homology to known proteins and are enriched in disordered regions and short linear interaction motifs. Published ribosome profiling data indicate translation of more than 100 novel sORFs, and mass spectrometry data provide evidence for more than 70 novel candidates.
  
  
  CONCLUSIONS
  Taken together, we identify hundreds of previously unknown conserved sORFs in major model organisms. Our computational analyses and integration with experimental data show that these sORFs are expressed, often translated, and sometimes widely conserved, in some cases even between vertebrates and invertebrates. We thus provide an integrated resource of putatively functional micropeptides for functional validation in vivo.
authors.affiliation: Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany. SDmackowiak@gmail.com.
authors.forename: Sebastian D
authors.initials: SD
authors.lastname: Mackowiak
authors.affiliation: Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany. henrik.zauber@mdc-berlin.de.
authors.forename: Henrik
authors.initials: H
authors.lastname: Zauber
authors.affiliation: Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany. chris.bielow@mdc-berlin.de.|Berlin Institute of Health, Kapelle-Ufer 2, 10117, Berlin, Germany. chris.bielow@mdc-berlin.de.
authors.forename: Chris
authors.initials: C
authors.lastname: Bielow
authors.affiliation: Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany. nisi_thiel@hotmail.de.
authors.forename: Denise
authors.initials: D
authors.lastname: Thiel
authors.affiliation: Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany. kamila.kutz@mdc-berlin.de.
authors.forename: Kamila
authors.initials: K
authors.lastname: Kutz
authors.affiliation: Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany. lorenzo.calviello@mdc-berlin.de.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Calviello
authors.affiliation: Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany. guido.mastrobuoni@mdc-berlin.de.
authors.forename: Guido
authors.initials: G
authors.lastname: Mastrobuoni
authors.affiliation: Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany. rajewsky@mdc-berlin.de.
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Rajewsky
authors.affiliation: Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany. stefan.kempa@mdc-berlin.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Kempa
authors.affiliation: Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany. matthias.selbach@mdc-berlin.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Selbach
authors.affiliation: Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany. benedikt.obermayer@mdc-berlin.de.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Obermayer
mesh_terms: D020413:3' Untranslated Regions; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D018388:Codon, Terminator; D017124:Conserved Sequence; D005091:Exons; D006801:Humans; D051379:Mice; D016366:Open Reading Frames; D010455:Peptides / Q000737:chemistry; D014176:Protein Biosynthesis; D016415:Sequence Alignment
chemical_list: D020413:3' Untranslated Regions; D018388:Codon, Terminator; D010455:Peptides