mRNA translational specialization by RBPMS presets the competence for cardiac commitment in hESCs.

The blueprints of developing organs are preset at the early stages of embryogenesis. Transcriptional and epigenetic mechanisms are proposed to preset developmental trajectories. However, we reveal that the competence for the future cardiac fate of human embryonic stem cells (hESCs) is preset in pluripotency by a specialized mRNA translation circuit controlled by RBPMS. RBPMS is recruited to active ribosomes in hESCs to control the translation of essential factors needed for cardiac commitment program, including Wingless/Integrated (WNT) signaling. Consequently, RBPMS loss specifically and severely impedes cardiac mesoderm specification, leading to patterning and morphogenetic defects in human cardiac organoids. Mechanistically, RBPMS specializes mRNA translation, selectively via 3'UTR binding and globally by promoting translation initiation. Accordingly, RBPMS loss causes translation initiation defects highlighted by aberrant retention of the EIF3 complex and depletion of EIF5A from mRNAs, thereby abrogating ribosome recruitment. We demonstrate how future fate trajectories are programmed during embryogenesis by specialized mRNA translation.

Phospho-RNA sequencing with circAID-p-seq.

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.

Active Ribosome Profiling with RiboLace: From Bench to Data Analysis.

Ribosome profiling is based on the deep sequencing of RNA fragments protected by ribosomes from nuclease digestion. This technique has been extensively used to study translation, with the unique ability to provide information about ribosomes positioning along transcripts at single-nucleotide resolution. Classical ribosome profiling approaches do not distinguish between fragments protected by either actively translating or inactive ribosomes. Here we describe an original method, called active ribosome profiling or RiboLace, which is based on a unique puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. This method allows reliable estimates of the translational state of any biological system, in high concordance with protein levels. RiboLace can be applied both in vitro and in vivo and generates snapshots of active ribosome footprints at single-nucleotide resolution and genome-wide level. RiboLace data are suitable for the analysis of translated genes, codon-specific translation rates, and local changes in ribosome occupancy profiles.

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 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.

SMN-primed ribosomes modulate the translation of transcripts related to spinal muscular atrophy.

The contribution of ribosome heterogeneity and ribosome-associated proteins to the molecular control of proteomes in health and disease remains unclear. Here, we demonstrate that survival motor neuron (SMN) protein-the loss of which causes the neuromuscular disease spinal muscular atrophy (SMA)-binds to ribosomes and that this interaction is tissue-dependent. SMN-primed ribosomes are preferentially positioned within the first five codons of a set of mRNAs that are enriched for translational enhancer sequences in the 5' untranslated region (UTR) and rare codons at the beginning of their coding sequence. These SMN-specific mRNAs are associated with neurogenesis, lipid metabolism, ubiquitination, chromatin regulation and translation. Loss of SMN induces ribosome depletion, especially at the beginning of the coding sequence of SMN-specific mRNAs, leading to impairment of proteins that are involved in motor neuron function and stability, including acetylcholinesterase. Thus, SMN plays a crucial role in the regulation of ribosome fluxes along mRNAs encoding proteins that are relevant to SMA pathogenesis.

Targeting Translation Activity at the Ribosome Interface with UV-Active Small 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.

Active Ribosome Profiling with RiboLace.

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.

Global translation variations in host cells upon attack of lytic and sublytic Staphylococcus aureus α-haemolysin.

Genome-wide analyses of translation can provide major contributions in our understanding of the complex interplay between virulent factors and host cells. So far, the activation of host translational control mechanisms by bacterial toxins, owing to specific recruitment of mRNAs, RNA-binding proteins (RBPs) and ncRNAs (non-coding RNAs), are far from being understood. In the present study, we characterize for the first time the changes experienced by the translational control system of host cells in response to the well-known Staphylococcus aureus α-haemolysin (AHL) under both sublytic and lytic conditions. By comparing variations occurring in the cellular transcriptome and translatome, we give evidence that global gene expression is primarily rewired at the translational level, with the contribution of the RBP ELAVL1 (HuR) in the sublytic response. These results reveal the importance of translational control during host-pathogen interaction, opening new approaches for AHL-induced diseases.

Detection of 3'-end RNA uridylation with a protein nanopore.

Post-transcriptional modifications of the 3'-ends of RNA molecules have a profound impact on their stability and processing in the cell. Uridylation, the addition of uridines to 3'-ends, has recently been found to be an important regulatory signal to stabilize the tagged molecules or to direct them toward degradation. Simple and cost-effective methods for the detection of this post-transcriptional modification are not yet available. Here, we demonstrate the selective and transient binding of 3'-uridylated ssRNAs inside the ß barrel of the staphylococcal α-hemolysin (αHL) nanopore and investigate the molecular basis of uridine recognition by the pore. We show the discrimination of 3'-oligouridine tails on the basis of their lengths and propose the αHL nanopore as a useful sensor for this biologically relevant RNA modification.