GSE167865
One-shot analysis of translated mammalian lncRNAs with AHARIBO
A vast portion of the mammalian genome is transcribed as long non-coding RNAs (lncRNAs) acting in the cytoplasm with largely unknown functions. Surprisingly, lncRNAs have been shown to interact with ribosomes, encode uncharacterized proteins, or act as ribosome sponges. These functions still remain mostly undetected and understudied owing to the lack of efficient tools for genome-wide simultaneous identification of ribosome-associated lncRNAs and peptide-producing lncRNAs. Here we present AHARIBO, a method for the detection of lncRNAs either untranslated, but associated with ribosomes, or encoding small peptides. Using AHARIBO in mouse embryonic stem cells during neuronal differentiation, we isolated ribosome-protected RNA fragments, translated RNAs and corresponding de novo synthesized polypeptides. Besides identifying mRNAs under active translation and associated ribosomes, we found and distinguished lncRNAs acting as ribosome sponges or encoding micropeptides, laying the ground for a better functional understanding of hundreds lncRNAs. Overall design: We performed standard and AHARIBO (AHA-mediated RIBOsome isolation) RIBO-Seq on mouse embryonic stem cells. AHARIBO RIBO-Seq is based on the isolation of ribosomes trapped with their nascent peptides, by incorporating the non-canonical amino acid L-azidohomoalanine (AHA) followed by ribosome profiling. Experiment were performed in biological triplicate (standard RIBO-Seq) and duplicate (AHARIBO RIBO-Seq).