Extensive identification and analysis of conserved small ORFs in animals

Abstract

BackgroundThere 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.ResultsHere, 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.ConclusionsTaken 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0742-x) contains supplementary material, which is available to authorized users.

The functions and unique features of long intergenic non-coding RNA

“…smORF regions are depleted of non-synonymous mutations and lack insertions and deletions, suggesting conservation at the peptide level 118,119 . Putative micropeptide-harbouring lincRNAs are expressed at higher levels and in a less restricted pattern than lincRNAs lacking smORFs 119 , implying that their translation or their peptide products may function more broadly than the transcription or transcripts of lincRNAs lacking smORFs. The most comprehensive smORF catalogue was derived from cross-species conservation-based in silico smORF predictions 119 .…”

New Peptides Under the s(ORF)ace of the Genome

“…Mackoviak et al [75] identified, computationally, a total of 2,002 novel putatively functional smORFs in 5 different organisms, based on their conservation patterns (obtained, briefly, with an SVM-based classifier, taking into account ORF conservation in multiple alignments, and PhyloCSF and PhastCons scores). These peptides map mostly to UTRs and LncRNAs, show little homology to known proteins, and are shorter than annotated smORFs, also having different aa sequence properties [75]. Interestingly these smORFs have Ribo-seq ORFscore values that are higher than non-coding controls, but lower than annotated smORFs.…”

“…In these organisms, HPLC-MS detects very few peptides from LncRNAs and uORFs (0%-0.3%), compared to annotated smORFs (12-33%), whereas Ribo-seq still detects a substantial amount of LncRNA smORFs and uORFs (3-30%, compared to 30-80% annotated smORFs), highlighting the difference in sensitivity between these techniques. The number of transcribed uORFs (*) was inferred from the number of transcripts with uORFs identified in other studies, for humans [61] and for zebrafish [29]; the number of peptides identified in humans by HPLC-MS ( †) were obtained from Mackowiak et al [75] B-The higher detection rates of annotated smORFs by HPLC-MS could be due to their higher levels of expression, and larger (and more stable) peptides, which also correlate with their closer resemblance to canonical proteins, in terms of functional signatures (protein domain content, conservation). Although these observations imply that annotated smORFs represent a functionally distinct class from LncRNA smORFs and uORFs, the identification of a growing number of biologically active peptides encoded in previously non-coding RNAs and uORFs (italics) proves that their functionality should not be systematically discarded.…”

Recognition of the polycistronic nature of human genes is critical to understanding the genotype-phenotype relationship

“…Moreover, some tools also predict the translation of alternative ORFs, such as SPECtre, All ORFs (≤100 codons) detected by ribosome profiling (Olexiouk et al 2016) N/A Identification of conserved small ORFs All predicted conserved small ORFs (>27 codons) in Supplementary Tables for five species (Mackowiak et al 2015) tsORFdb Theoretical short ORF database Systematic six-frame translation, using ATG and non-ATG initiation codons (Heo et al 2010) (Hao et al 2017) www.genome.org…”