Long noncoding RNAs are rarely translated in two human cell lines

Data from the Encyclopedia of DNA Elements (ENCODE) project show over 9640 human genome loci classified as long noncoding RNAs (IncRNAs), yet only 100 have been deeply characterized to determine their role in the cell. To measure the protein-coding output from these RNAs, we jointly analyzed two recent data sets produced in the ENCODE project: tandem mass spectrometry (MS/MS) data mapping expressed peptides to their encoding genomic loci, and RNA-seq data generated by ENCODE in long polyA+ and polyA- fractions in the cell lines K562 and GMI2878. We used the machine-learning algorithm RuleFit3 to regress the peptide data against RNA expression data. The most important covariate for predicting translation was, surprisingly, the Cytosol polyA- fraction in both cell lines. LncRNAs are similar to 13-fold less likely to produce detectable peptides than similar mRNAs, indicating that similar to 92% of GENCODE v7 IncRNAs are not translated in these two ENCODE cell lines. Intersecting 9640 IncRNA loci with 79,333 peptides yielded 85 unique peptides matching 69 IncRNAs. Most cases were due to a coding transcript misannotated as IncRNA. Two exceptions were an unprocessed pseudogene and a bona fide IncRNA gene, both with open reading frames (ORFs) compromised by upstream stop codons. All potentially translatable IncRNA ORFs had only a single peptide match, indicating low protein abundance and/or false-positive peptide matches. We conclude that with very few exceptions, ribosomes are able to distinguish coding from noncoding transcripts and, hence, that ectopic translation and cryptic mRNAs are rare in the human IncRNAome.