Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.

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 peptides, 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 and peptide-producing lncRNAs. Here, we present AHA-mediated RIBOsome isolation (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 peptides. 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 of lncRNAs.

Most RNA footprinting approaches that require ribonuclease cleavage generate RNA fragments bearing a phosphate or cyclic phosphate group at their 3′ end. Unfortunately, current library preparation protocols rely only on a 3′ hydroxyl group for adaptor ligation or poly-A tailing. Here, we developed circAID-p-seq, a PCR-free library preparation for selective 3′ phospho-RNA sequencing. As a proof of concept, we applied circAID-p-seq to ribosome profiling, which is based on sequencing of RNA fragments protected by ribosomes after endonuclease digestion. CircAID-p-seq, combined with the dedicated computational pipeline circAidMe, facilitates accurate, fast and highly efficient sequencing of phospho-RNA fragments from eukaryotic cells and tissues. We used circAID-p-seq to portray ribosome occupancy in transcripts, providing a versatile and PCR-free strategy to possibly unravel any endogenous 3′-phospho RNA molecules.

Puromycin is a well-known antibiotic that is used to study the mechanism of protein synthesis and to monitor ribosome activity due to its incorporation into nascent peptide chains. However, puromycin effects outside the ribosome catalytic core remain unexplored. Here, we developed two analogues (3PB and 3PC) of the 3′-end of tyrosylated-tRNA that can efficiently interact with several proteins associated with ribosomes. We biochemically characterized the binding of these analogues and globally mapped the direct small molecule–protein interactions in living cells using clickable and photoreactive puromycin-like probes in combination with in-depth mass spectrometry. We identified a list of proteins targeted by the molecules during ribosome activity (e.g., GRP78), and we addressed possible uses of the probes to sense the activity of protein synthesis and to capture associated RNA. By coupling genome-wide RNA sequencing methods with these molecules, the characterization of unexplored translational control mechanisms will be feasible.

Gene regulation through translation is critical for spatiotemporal protein expression. Internal ribosomal entry sites (IRESes) mediate mRNA-specific translation by recruiting ribosomes to 5′ untranslated regions. Circular RNAs (circRNAs), naturally occurring and stable RNAs, are increasingly used as synthetic tools for sustained therapeutic protein translation by IRES-driven initiation. However, the functionality of different IRESes in synthetic circRNAs remains sparsely characterized. We systematically examine circRNA reporter translation by viral and cellular IRESes in human cells and in diverse in vitro translation systems. Improved circRNA purification by urea-PAGE and RNase R-treatment removes contaminants that induce RNA sensing. Viral CVB3 and HCV, as well as cellular Hoxa9, Chrdl1, Cofilin and c-Myc IRESes, effectively drive circRNA translation. We also establish circRNA translation in a recently developed human cell-free extract that recapitulates IRES-dependent regulation, and allows for precise engineering of HCV IRES-mediated translation. These findings inform IRES selection for synthetic circRNA translation relevant for circRNA-based medicine. GRAPHICAL