Computational identification of extra coding RNAs related to HIV1 infection A few studies suggest that many protein coding loci also transcribe extracoding RNAs (ecRNAs), a distinct class of long noncoding RNAs. These ecRNAs span over and beyond mRNAs originating from the same loci, are much less abundant than mRNAs, and lack polyadenylation. Previously, ecRNAs were found to regulate the transcription of mRNAs from the same loci epigenetically. However, further investigation of ecRNAs is impeded by the lack of accurate ecRNA annotation and quantification. Here, to uncover these mRNAoverlapping ecRNAs, we first depleted polyadenylated RNAs, i.e. mRNAs, from total RNAs and performed deep sequencing analysis of the remaining total RNAs. Next, we devised a computational strategy to identify ecRNA transcribing loci from this unique sequencing data. Our approach uses a combination of features including the ratio of intronic to exonic read counts, the percent read coverage of introns, exons, and flanking regions, and the ratio of flanking to intronic read counts. In this pilot study of HIV1 infected CD4 T cells, we observed transcriptional signatures of ecRNAs from 880 (xe2x88xbc11%) of 7,690 expressed coding genes. We expect these custom computational developments will enable a better understanding of the involvement of ecRNAs in regulating host response to HIV infection and in other conditions.

Ribosomal inframe mistranslation of stop codons in multiple open reading frames of specific human long noncoding RNAs. One of the major discoveries of the early postgenomic era, as embodied by the gene catalogs of the FANTOM and ENCODE (Encyclopedia of DNA Elements) consortia, is that twothirds of human genes do not encode known proteins. Those 40,000 nonproteincoding (noncoding RNA) human genes (www.gencodegenes.org) remain poorly understood. Long noncoding RNA (lncRNA) genes are the most numerous category of human ncRNA genes. Hundreds of lncRNAs have recentlydiscovered functions and are now understood to be fundamental nuclear and cytoplasmic, epigenetic and posttranscriptional, positive and negative regulators of gene expression in normal cellular functions and in a wide range of human diseases. However, the functions, if any, of the vast majority of lncRNAs remain obscure. Significantly, an unconventional role for their transcripts as unexpected defacto messenger RNAs has not been formally excluded. Ribosome profiling (Riboseq) predicts translational potential; nonetheless, without independent evidence of proteins matching lncRNA open reading frames (ORFs), ribosome binding does not prove translation. We were the first to massspectrometrically document translation of specific lncRNAs in human cells (https:genome.cshlp.orgcontent2291646.long). We have now performed a global search for lncRNA translation in human MCF7 breast cancer cells, integrating strandspecific RNAseq, Riboseq, and deep mass spectrometry of trypsindigested u003c15kDa fractions postUHPLC (Orbitrap MSMS) in biological quadruplicates by two independent core facilities. We excluded knownprotein matches. UCSC Genome Browserassisted manual annotation of imperfect (trypticdigestpeptides)to(lncRNAthreeframe translations) alignments initially revealed three peptides hypothetically explicable by xe2x80x9cstoptononstopxe2x80x9d in frame replacement of stop codons by amino acids in two ORFs of the lncRNA MMP24AS1. To search for this phenomenon genomewide, we designed and implemented an unprecedented computational pipeline, matching trypticdigest spectra to wildcardinsteadofstop versions of repeatmasked, sixframe, wholegenome translations. Along with singleton stoptononstop events affecting four other lncRNAs, we identified 24 additional peptides with stoptononstop inframe substitutions from multiple MMP24AS1 lncRNA ORFs. Only UAG and UGA, but not UAA, stop codons were affected. All MMP24AS1matching spectra met the same significance thresholds as highconfidence knownprotein signatures. Targeted resequencing of MMP24AS1 genomic DNA and cDNA from the same samples did not reveal any mutations, polymorphisms, or sequencingdetectable RNA editing. We have therefore discovered an apparent genespecific violation of the genetic code. It highlights the importance of matching peptides to wholegenome, not knowngenesonly, ORFs in massspectrometry workflows, and suggests a new mechanism enhancing the combinatorial complexity of the proteome.