Translation and Natural Selection of long non-canonical RNA micropeptides

Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides but lacking canonical coding sequences. Apparently unable to produce peptides, lncRNA function seems to only involve RNA sequence and structure. Here, we exhaustively detect in-vivo translation of small open reading frames (small ORFs) within lncRNAs using Ribosomal profiling during Drosophila melanogaster embryogenesis. We show that around 30% of lncRNAs contain small ORFs engaged by ribosomes, leading to regulated translation of 100 to 300 micropeptides. We identify lncRNA features that favour translation, such as cistronicity, Kozak sequences, and conservation. For this latter, we develop a bioinformatics pipeline to detect small ORF homologues, and we reveal evidence of natural selection favouring the conservation of micropeptide sequence and function across evolution. Our results expand the repertoire of lncRNA functions, and suggest that lncRNAs give rise to novel coding genes throughout evolution. Since most lncRNAs contain small ORFs with as yet unknown translation potential, we propose to rename them “long non-canonical RNAs”. Overall design: We performed gene expression profiling analysis by additional sequencing of previously-obtained Ribo-Seq and RNA-Seq libraries (Embryo and S2 cells), see Patraquim et a. (2020). Comparative gene expression profiling analysis of RNA-seq data for C3H10T1/2 cells and its KD derivatives (shKDM4D, shNFIB, and shMLL1).