Transparent Process

Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation

Ariel A Bazzini, Timothy G Johnstone, Romain Christiano, Sebastian D Mackowiak, Benedikt Obermayer, Elizabeth S Fleming, Charles E Vejnar, Miler T Lee, Nikolaus Rajewsky, Tobias C Walther, Antonio J Giraldez

Author Affiliations

  1. Ariel A Bazzini*,1,,
  2. Timothy G Johnstone1,,
  3. Romain Christiano2,
  4. Sebastian D Mackowiak3,
  5. Benedikt Obermayer3,
  6. Elizabeth S Fleming1,
  7. Charles E Vejnar1,
  8. Miler T Lee1,
  9. Nikolaus Rajewsky*,3,
  10. Tobias C Walther2 and
  11. Antonio J Giraldez*,1,4
  1. 1Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
  2. 2Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA
  3. 3Systems Biology of Gene Regulatory Elements, Max‐Delbrück‐Center for Molecular Medicine, Berlin, Germany
  4. 4Yale Stem Cell Center Yale University School of Medicine, New Haven, CT, USA
  1. * Corresponding author. Tel: +1 203 785 5450; Fax: +1 203 785 4415; E‐mail: ariel.bazzini{at}

    Corresponding author. Tel: +49 30 9406 2999; Fax: +49 30 9406 3068; E‐mail: rajewsky{at}

    Corresponding author. Tel: +1 203 785 5423; Fax: +1 203 785 4415; E‐mail: antonio.giraldez{at}

View Full Text


Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide‐encoding genes in vertebrates, providing an entry point to define their function in vivo.


Embedded Image

The combination of ORFscore and micPDP enable high confidence prediction of many, small translated ORFs that were functionally not appreciated or previously annotated as lincRNAs.

  • “ORFScore” explores high‐resolution footprinting with ribosome phasing to identify novel, translated micropeptides in genes previously thought to lack coding potential.

  • “micPDP” is a new computational pipeline that identifies micropeptides under negative selection across species.

  • The combination of these techniques enabled high confidence prediction of numerous small translated ORFs suitable for functional characterization.

  • Received March 6, 2014.
  • Revision received March 13, 2014.
  • Accepted March 14, 2014.
View Full Text

Subscribers, please sign in with your username and password.

Log in through your institution