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SummaryDespite predicted to lack coding potential, cytoplasmic long non-coding (lnc)RNAs can associate with ribosomes, resulting in some cases into the production of functional peptides. However, the biological and mechanistic relevance of this pervasive lncRNAs translation remains poorly studied. In yeast, cytoplasmic Xrn1-sensitive lncRNAs (XUTs) are targeted by the Nonsense-Mediated mRNA Decay (NMD), suggesting a translation-dependent degradation process. Here, we report that XUTs are translated, which impacts their abundance. We show that XUTs globally accumulate upon translation elongation inhibition, but not when initial ribosome loading is impaired. Translation also affects XUTs independently of NMD, by interfering with their decapping. Ribo-Seq confirmed ribosomes binding to XUTs and identified actively translated small ORFs in their 5’-proximal region. Mechanistic analyses revealed that their NMD-sensitivity depends on the 3’-untranslated region length. Finally, we detected the peptide derived from the translation of an NMD-sensitive XUT reporter in NMD-competent cells. Our work highlights the role of translation in the metabolism of XUTs, which could contribute to expose genetic novelty to the natural selection, while NMD restricts their expression.

Human immunodeficiency virus type-1 (HIV-1) is a complex retrovirus which relies on alternative splicing, translational and post-translational mechanisms to produce more than 15 functional proteins from its single ∼10kb transcriptional unit. Here, we have applied ribosome profiling and nascent protein labeling at different time points during infection of CD4+ T lymphocytes to characterize the translational landscape of cellular and viral transcripts during the course of infection. Our results indicate a strong impact of viral infection on host cellular transcript levels but a modest impact on global translation rates. Analysis of ribosome profiling reads from viral transcripts reveals extensive and productive non-AUG translation of small peptides from multiple upstream open reading-frames (uORFs) located in the 5’ long terminal repeat. Remarkably, these uORFs derived peptides elicit specific T cell responses in HIV-infected individuals. uORFs are conserved among other retroviruses and, together with the TAR sequence, condition the dependency on DDX3 for efficient translation of the main viral open-reading frames.

Premature termination codons (PTCs) account for 10% to 20% of genetic diseases in humans. The gene inactivation resulting from PTC can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a new reporter cell line and performed high-throughput screening (HTS) to identify potential new readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy. We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of natural stop codons.

The noncoding genome plays an important role in de novo gene birth and in the emergence of genetic novelty. Nevertheless, how noncoding sequences’ properties could promote the birth of novel genes and shape the evolution and the structural diversity of proteins remains unclear. Therefore, by combining different bioinformatic approaches, we characterized the fold potential diversity of the amino acid sequences encoded by all intergenic ORFs (Open Reading Frames) of S. cerevisiae with the aim of (i) exploring whether the large structural diversity observed in proteomes is already present in noncoding sequences, and (ii) estimating the potential of the noncoding genome to produce novel protein bricks that can either give rise to novel genes or be integrated into pre-existing proteins, thus participating in protein structure diversity and evolution. We showed that amino acid sequences encoded by most yeast intergenic ORFs contain the elementary building blocks of protein structures. Moreover, they encompass the large structural diversity of canonical proteins with strikingly the majority predicted as foldable. Then, we investigated the early stages of de novo gene birth by identifying intergenic ORFs with a strong translation signal in ribosome profiling experiments and by reconstructing the ancestral sequences of 70 yeast de novo genes. This enabled us to highlight sequence and structural factors determining de novo gene emergence. Finally, we showed a strong correlation between the fold potential of de novo proteins and the one of their ancestral amino acid sequences, reflecting the relationship between the noncoding genome and the protein structure universe.

Little is known about the rate of emergence of genes de novo, how they spread in populations and what their initial properties are. We examined wild Saccharomyces paradoxus populations to characterize the diversity and turnover of intergenic ORFs over short evolutionary time-scales. We identified ~34,000 intergenic ORFs per individual genome for a total of ~64,000 orthogroups, which resulted from an estimated turnover rate relatively smaller than the rate of gene duplication in yeast. Hundreds of intergenic ORFs show translation signatures, similar to canonical genes, but lower translation efficiency, which could reduce their potential production cost or simply reflect a lack of optimization. Translated intergenic ORFs tend to display low expression levels with sequence properties that are on average closer to expectations based on intergenic sequences. However, some predicted de novo polypeptides with gene-like properties emerged from ancient as well as recent birth events, illustrating that the raw material for functional innovations may appear even over short evolutionary time-scales. Our results suggest that variation in the mutation rate along the genome impacts the turnover of random polypeptides, which may in turn influence their early evolutionary trajectory. Whereas low mutation rate regions allow more time for random intergenic ORFs to evolve and become functional before being lost, mutation hotspots allow for the rapid exploration of the molecular landscape, thereby increasing the probability to acquire a polypeptide with immediate gene-like properties and thus functional potential.

Sequences in certain mRNAs program the ribosome to undergo a noncanonical translation event, translational frameshifting, translational hopping, or termination readthrough. These sequences are termed recoding sites, because they cause the ribosome to change temporarily its coding rules. Cis and trans-acting factors sensitively modulate the efficiency of recoding events. In an attempt to quantitate the effect of these factors we have developed a dual-reporter vector using the lacZ and luc genes to directly measure recoding efficiency. We were able to confirm the effect of several factors that modulate frameshift or readthrough efficiency at a variety of sites. Surprisingly, we were not able to confirm that the complex of factors termed the surveillance complex regulates translational frameshifting. This complex regulates degradation of nonsense codon-containing mRNAs and we confirm that it also affects the efficiency of nonsense suppression. Our data suggest that the surveillance complex is not a general regulator of translational accuracy, but that its role is closely tied to the translational termination and initiation processes.

The complexity of the nonsense-mediated decay (NMD) system makes it difficult to study by comparing the expression of various single reporter constructs. The known effects of the NMD genes include a reduction both in mRNA stability (reviewed by Czaplinski et al., 1999) and in the efficiency of translational initiation (Muhlrad & Parker, 1999) of nonsense-containing plasmids as well as an apparent increase in the efficiency of translational termination as evidenced by increased readthrough of nonsense mutations (Bidou et al., 2000; Maderazo et al., 2000). The single reporter system can not distinguish among these effects and inference is required to determine which mechanism underlies any observed phenotypic effect on gene expression. It is particularly problematic to differentiate the effects of translation initiation accuracy from putative effects on translational frameshifting. The dual reporter system used in our work isolates the effect of translational frameshifting from effects on mRNA stability, initiation or termination. Much is made by Dinman et al. of the relative effects of various mutations, yet it remains unclear whether these are fundamental differences or simply differences in phenotypic strength of the various mutations.