Abstract 189: DWORF: Discovery and Characterization of a Cardiac Micropeptide Encoded in a Putative Long Noncoding RNA
Background: Mammalian transcriptomes contain thousands of potential open reading frames (ORFs) with fewer than fifty codons. Most of these ORFs are presumed to be random and therefore noncoding, but recently several genes annotated as long noncoding RNAs (lncRNAs) were shown to encode functional micropeptides. Objective: Identify and characterize novel micropeptides encoded in cardiac-expressed lncRNAs using comparative genomics. Methods and Results: Using codon substitution frequency, a comparative genomics strategy, we identified several previously unknown small proteins, including a 34-amino acid micropeptide that we have named DWORF (Dwarf Open Reading Frame). DWORF is a tail-anchored transmembrane protein that localizes to the sarcoplasmic reticulum and is expressed only in cardiac ventricle and slow-twitch skeletal muscle by quantitative PCR. We subsequently derived a custom antibody targeting the N-terminal residues of DWORF. Western blots using this antibody revealed a 4 kDa protein that is expressed in cardiac ventricle and soleus muscle, but not other tissues. We further validated specificity of this signal by disrupting the reading frame of DWORF in mice using CRISPR/Cas9 mutagenesis, conclusively demonstrating that the mRNA encodes a small protein. Transgenic overexpression of DWORF in cardiac myocytes resulted in increased peak amplitude of Ca2+ transients and improved Ca2+ resequestration kinetics with no apparent adverse effects. These findings suggest that DWORF specifically enhances activity of the sarco/endoplasmic reticulum calcium ATPase (SERCA). Conclusions: Some putative lncRNAs encode micropeptides that can be identified using bioinformatics strategies. Micropeptides, though likely too small to have enzymatic activity of their own, may have modulatory functions in important cellular processes such as calcium handling, as we have demonstrated in the case of the muscle-restricted micropeptide DWORF.
Author Disclosures: B.R. Nelson: None. C.A. Makarewich: None. A.L. Reese: None. B.R. Winders: None. D.M. Anderson: None. J.R. McAnally: None. E.T. Kavalali: None. R. Bassel-Duby: None. E.N. Olson: None.
- © 2015 by American Heart Association, Inc.