Critical cis-parameters influence STructure assisted RNA translation (START) initiation on non-AUG codons in eukaryotes.

In eukaryotes, translation initiation is a highly regulated process, which combines cis-regulatory sequences located on the messenger RNA along with trans-acting factors like eukaryotic initiation factors (eIF). One critical step of translation initiation is the start codon recognition by the scanning 43S particle, which leads to ribosome assembly and protein synthesis. In this study, we investigated the involvement of secondary structures downstream the initiation codon in the so-called START (STructure-Assisted RNA translation) mechanism on AUG and non-AUG translation initiation. The results demonstrate that downstream secondary structures can efficiently promote non-AUG translation initiation if they are sufficiently stable to stall a scanning 43S particle and if they are located at an optimal distance from non-AUG codons to stabilize the codon-anticodon base pairing in the P site. The required stability of the downstream structure for efficient translation initiation varies in distinct cell types. We extended this study to genome-wide analysis of functionally characterized alternative translation initiation sites in Homo sapiens. This analysis revealed that about 25% of these sites have an optimally located downstream secondary structure of adequate stability which could elicit START, regardless of the start codon. We validated the impact of these structures on translation initiation for several selected uORFs.

Purification of In Vivo or In Vitro-Assembled RNA-Protein Complexes by RNA Centric Methods.

Throughout their entire life cycle, RNAs are associated with RNA-binding proteins (RBPs), forming ribonucleoprotein (RNP) complexes with highly dynamic compositions and very diverse functions in RNA metabolism, including splicing, translational regulation, ribosome assembly. Many RNPs remain poorly characterized due to the challenges inherent in their purification and subsequent biochemical characterization. Therefore, developing methods to isolate specific RNA-protein complexes is an important initial step toward understanding their function. Many elegant methodologies have been developed to isolate RNPs. This chapter describes different approaches and methods devised for RNA-specific purification of a target RNP. We focused on general methods for selecting RNPs that target a given RNA under conditions favourable for the copurification of associated factors including RNAs and protein components of the RNP.

Immunoprecipitation Methods to Isolate Messenger Ribonucleoprotein Complexes (mRNP).

Throughout their life cycle, messenger RNAs (mRNAs) associate with proteins to form ribonucleoproteins (mRNPs). Each mRNA is part of multiple successive mRNP complexes that participate in their biogenesis, cellular localization, translation and decay. The dynamic composition of mRNP complexes and their structural remodelling play crucial roles in the control of gene expression. Studying the endogenous composition of different mRNP complexes is a major challenge. In this chapter, we describe the variety of protein-centric immunoprecipitation methods available for the identification of mRNP complexes and the requirements for their experimental settings.

Pathophysiology of human mitochondrial tRNA metabolism.

Mitochondria play multiple critical roles in cellular activity. In particular, mitochondrial translation is pivotal in the regulation of mitochondrial and cellular homeostasis. In this forum article, we discuss human mitochondrial tRNA metabolism and highlight its tight connection with various mitochondrial diseases caused by mutations in aminoacyl-tRNA synthetases, tRNAs, and tRNA-modifying enzymes.

Mammalian mitochondrial translation infidelity leads to oxidative stress-induced cell cycle arrest and cardiomyopathy.

Proofreading (editing) of mischarged tRNAs by cytoplasmic aminoacyl-tRNA synthetases (aaRSs), whose impairment causes neurodegeneration and cardiac diseases, is of high significance for protein homeostasis. However, whether mitochondrial translation needs fidelity and the significance of editing by mitochondrial aaRSs have been unclear. Here, we show that mammalian cells critically depended on the editing of mitochondrial threonyl-tRNA synthetase (mtThrRS, encoded by Tars2), disruption of which accumulated Ser-tRNAThr and generated a large abundance of Thr-to-Ser misincorporated peptides in vivo. Such infidelity impaired mitochondrial translation and oxidative phosphorylation, causing oxidative stress and cell cycle arrest in the G0/G1 phase. Notably, reactive oxygen species (ROS) scavenging by N-acetylcysteine attenuated this abnormal cell proliferation. A mouse model of heart-specific defective mtThrRS editing was established. Increased ROS levels, blocked cardiomyocyte proliferation, contractile dysfunction, dilated cardiomyopathy, and cardiac fibrosis were observed. Our results elucidate that mitochondria critically require a high level of translational accuracy at Thr codons and highlight the cellular dysfunctions and imbalance in tissue homeostasis caused by mitochondrial mistranslation.

SARS-CoV-2 NSP1 induces mRNA cleavages on the ribosome.

In severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the non-structural protein NSP1 inhibits translation of host mRNAs by binding to the mRNA entry channel of the ribosome and, together with the 5'-untranslated region (UTR) of the viral mRNAs, allows the evasion of that inhibition. Here, we show that NSP1 mediates endonucleolytic cleavages of both host and viral mRNAs in the 5'UTR, but with different cleavage patterns. The first pattern is observed in host mRNAs with cleavages interspersed regularly and close to the 5' cap (6-11 nt downstream of the cap). Those cleavage positions depend more on the position relative to the 5' cap than on the sequence itself. The second cleavage pattern occurs at high NSP1 concentrations and only in SARS-CoV-2 RNAs, with the cleavages clustered at positions 45, 46 and 49. Both patterns of cleavage occur with the mRNA and NSP1 bound to the ribosome, with the SL1 hairpin at the 5' end sufficient to protect from NSP1-mediated degradation at low NSP1 concentrations. We show further that the N-terminal domain of NSP1 is necessary and sufficient for efficient cleavage. We suggest that in the ribosome-bound NSP1 protein the catalytic residues of the N-terminal domain are unmasked by the remodelling of the α1- and α2-helices of the C-terminal domain.

The tRNA identity landscape for aminoacylation and beyond.

tRNAs are key partners in ribosome-dependent protein synthesis. This process is highly dependent on the fidelity of tRNA aminoacylation by aminoacyl-tRNA synthetases and relies primarily on sets of identities within tRNA molecules composed of determinants and antideterminants preventing mischarging by non-cognate synthetases. Such identity sets were discovered in the tRNAs of a few model organisms, and their properties were generalized as universal identity rules. Since then, the panel of identity elements governing the accuracy of tRNA aminoacylation has expanded considerably, but the increasing number of reported functional idiosyncrasies has led to some confusion. In parallel, the description of other processes involving tRNAs, often well beyond aminoacylation, has progressed considerably, greatly expanding their interactome and uncovering multiple novel identities on the same tRNA molecule. This review highlights key findings on the mechanistics and evolution of tRNA and tRNA-like identities. In addition, new methods and their results for searching sets of multiple identities on a single tRNA are discussed. Taken together, this knowledge shows that a comprehensive understanding of the functional role of individual and collective nucleotide identity sets in tRNA molecules is needed for medical, biotechnological and other applications.

eIF3 Interacts with Selenoprotein mRNAs.

The synthesis of selenoproteins requires the co-translational recoding of an in-frame UGASec codon. Interactions between the Selenocysteine Insertion Sequence (SECIS) and the SECIS binding protein 2 (SBP2) in the 3'untranslated region (3'UTR) of selenoprotein mRNAs enable the recruitment of the selenocysteine insertion machinery. Several selenoprotein mRNAs undergo unusual cap hypermethylation and are not recognized by the translation initiation factor 4E (eIF4E) but nevertheless translated. The human eukaryotic translation initiation factor 3 (eIF3), composed of 13 subunits (a-m), can selectively recruit several cellular mRNAs and plays roles in specialized translation initiation. Here, we analyzed the ability of eIF3 to interact with selenoprotein mRNAs. By combining ribonucleoprotein immunoprecipitation (RNP IP) in vivo and in vitro with cross-linking experiments, we found interactions between eIF3 and a subgroup of selenoprotein mRNAs. We showed that eIF3 preferentially interacts with hypermethylated capped selenoprotein mRNAs rather than m7G-capped mRNAs. We identified direct contacts between GPx1 mRNA and eIF3 c, d, and e subunits and showed the existence of common interaction patterns for all hypermethylated capped selenoprotein mRNAs. Differential interactions of eIF3 with selenoprotein mRNAs may trigger specific translation pathways independent of eIF4E. eIF3 could represent a new player in the translation regulation and hierarchy of selenoprotein expression.

Viral and cellular translation during SARS-CoV-2 infection.

SARS-CoV-2 is a betacoronavirus that emerged in China in December 2019 and which is the causative agent of the Covid-19 pandemic. This enveloped virus contains a large positive-sense single-stranded RNA genome. In this review, we summarize the current knowledge on the molecular mechanisms for the translation of both viral transcripts and cellular messenger RNAs. Non-structural proteins are encoded by the genomic RNA and are produced in the early steps of infection. In contrast, the structural proteins are produced from subgenomic RNAs that are translated in the late phase of the infectious program. Non-structural protein 1 (NSP1) is a key molecule that regulates both viral and cellular translation. In addition, NSP1 interferes with multiple steps of the interferon I pathway and thereby blocks host antiviral responses. Therefore, NSP1 is a drug target of choice for the development of antiviral therapies.

Correlated sequence signatures are present within the genomic 5'UTR RNA and NSP1 protein in coronaviruses.

The 5'UTR part of coronavirus genomes plays key roles in the viral replication cycle and translation of viral mRNAs. The first 75-80 nt, also called the leader sequence, are identical for genomic mRNA and subgenomic mRNAs. Recently, it was shown that cooperative actions of a 5'UTR segment and the nonstructural protein NSP1 are essential for both the inhibition of host mRNAs and for specific translation of viral mRNAs. Here, sequence analyses of both the 5'UTR RNA segment and the NSP1 protein have been done for several coronaviruses, with special attention to the betacoronaviruses. The conclusions are: (i) precise specific molecular signatures can be found in both the RNA and the NSP1 protein; (ii) both types of signatures correlate between each other. Indeed, definite sequence motifs in the RNA correlate with sequence motifs in the protein, indicating a coevolution between the 5'UTR and NSP1 in betacoronaviruses. Experimental mutational data on 5'UTR and NSP1 from SARS-CoV-2 using cell-free translation extracts support these conclusions and show that some conserved key residues in the amino-terminal half of the NSP1 protein are essential for evasion to the inhibitory effect of NSP1 on translation.

Translation inhibitory elements from Hoxa3 and Hoxa11 mRNAs use uORFs for translation inhibition.

During embryogenesis, Hox mRNA translation is tightly regulated by a sophisticated molecular mechanism that combines two RNA regulons located in their 5'UTR. First, an internal ribosome entry site (IRES) enables cap-independent translation. The second regulon is a translation inhibitory element or TIE, which ensures concomitant cap-dependent translation inhibition. In this study, we deciphered the molecular mechanisms of mouse Hoxa3 and Hoxa11 TIEs. Both TIEs possess an upstream open reading frame (uORF) that is critical to inhibit cap-dependent translation. However, the molecular mechanisms used are different. In Hoxa3 TIE, we identify an uORF which inhibits cap-dependent translation and we show the requirement of the non-canonical initiation factor eIF2D for this process. The mode of action of Hoxa11 TIE is different, it also contains an uORF but it is a minimal uORF formed by an uAUG followed immediately by a stop codon, namely a 'start-stop'. The 'start-stop' sequence is species-specific and in mice, is located upstream of a highly stable stem loop structure which stalls the 80S ribosome and thereby inhibits cap-dependent translation of Hoxa11 main ORF.

RNA Secondary Structure Study by Chemical Probing Methods Using DMS and CMCT.

RNA folds into secondary structures that can serve in understanding various RNA functions (Weeks KM. Curr Opin Struct Biol 20(3):295-304, 2010). Chemical probing is a method that enables the characterization of RNA secondary structures using chemical reagents that specifically modify RNA nucleotides that are located in single-stranded areas. In our protocol, we used Dimethyl Sulfate (DMS) and Cyclohexyl-3-(2-Morpholinoethyl) Carbodiimide metho-p-Toluene sulfonate (CMCT) that are both base-specific modifying reagents (Behm-Ansmant I, et al. J Nucleic Acids 2011:408053, 2011). These modifications are mapped by primer extension arrests using 5' fluorescently labeled primers. In this protocol, we show a comprehensive method to identify RNA secondary structures in vitro using fluorescently labeled oligos. To demonstrate the efficiency of the method, we give an example of a structure we have designed which corresponds to a part of the 5'-UTR regulatory element called Translation Inhibitory Element (TIE) from Hox a3 mRNA (Xue S, et al. Nature 517(7532):33-38, 2015).

eIF3 interacts with histone H4 messenger RNA to regulate its translation.

In eukaryotes, various alternative translation initiation mechanisms have been unveiled for the translation of specific mRNAs. Some do not conform to the conventional scanning-initiation model. Translation initiation of histone H4 mRNA combines both canonical (cap-dependent) and viral initiation strategies (no-scanning, internal recruitment of initiation factors). Specific H4 mRNA structures tether the translation machinery directly onto the initiation codon and allow massive production of histone H4 during the S phase of the cell cycle. The human eukaryotic translation initiation factor 3 (eIF3), composed of 13 subunits (a-m), was shown to selectively recruit and control the expression of several cellular mRNAs. Whether eIF3 mediates H4 mRNA translation remains to be elucidated. Here, we report that eIF3 binds to a stem-loop structure (eIF3-BS) located in the coding region of H4 mRNA. Combining cross-linking and ribonucleoprotein immunoprecipitation experiments in vivo and in vitro, we also found that eIF3 binds to H1, H2A, H2B, and H3 histone mRNAs. We identified direct contacts between eIF3c, d, e, g subunits, and histone mRNAs but observed distinct interaction patterns to each histone mRNA. Our results show that eIF3 depletion in vivo reduces histone mRNA binding and modulates histone neosynthesis, suggesting that synthesis of histones is sensitive to the levels of eIF3. Thus, we provide evidence that eIF3 acts as a regulator of histone translation.

Distinct pathogenic mechanisms of various RARS1 mutations in Pelizaeus-Merzbacher-like disease.

Mutations of the genes encoding aminoacyl-tRNA synthetases are highly associated with various central nervous system disorders. Recurrent mutations, including c.5A>G, p.D2G; c.1367C>T, p.S456L; c.1535G>A, p.R512Q and c.1846_1847del, p. Y616Lfs*6 of RARS1 gene, which encodes two forms of human cytoplasmic arginyl-tRNA synthetase (hArgRS), are linked to Pelizaeus-Merzbacher-like disease (PMLD) with unclear pathogenesis. Among these mutations, c.5A>G is the most extensively reported mutation, leading to a p.D2G mutation in the N-terminal extension of the long-form hArgRS. Here, we showed the detrimental effects of R512Q substitution and ΔC mutations on the structure and function of hArgRS, while the most frequent mutation c.5A>G, p.D2G acted in a different manner without impairing hArgRS activity. The nucleotide substitution c.5A>G reduced translation of hArgRS mRNA, and an upstream open reading frame contributed to the suppressed translation of the downstream main ORF. Taken together, our results elucidated distinct pathogenic mechanisms of various RARS1 mutations in PMLD.

Secondary structure of the SARS-CoV-2 5'-UTR.

The SARS-CoV-2, a positive-sense single-stranded RNA Coronavirus, is a global threat to human health. Thus, understanding its life cycle mechanistically would be important to facilitate the design of antiviral drugs. A key aspect of viral progression is the synthesis of viral proteins by the ribosome of the human host. In Coronaviruses, this process is regulated by the viral 5' and 3' untranslated regions (UTRs), but the precise regulatory mechanism has not yet been well understood. In particular, the 5'-UTR of the viral genome is most likely involved in translation initiation of viral proteins. Here, we performed inline probing and RNase V1 probing to establish a model of the secondary structure of SARS-CoV-2 5'-UTR. We found that the 5'-UTR contains stable structures including a very stable four-way junction close to the AUG start codon. Sequence alignment analysis of SARS-CoV-2 variants 5'-UTRs revealed a highly conserved structure with few co-variations that confirmed our secondary structure model based on probing experiments.

The viral protein NSP1 acts as a ribosome gatekeeper for shutting down host translation and fostering SARS-CoV-2 translation.

SARS-CoV-2 coronavirus is responsible for Covid-19 pandemic. In the early phase of infection, the single-strand positive RNA genome is translated into non-structural proteins (NSP). One of the first proteins produced during viral infection, NSP1, binds to the host ribosome and blocks the mRNA entry channel. This triggers translation inhibition of cellular translation. In spite of the presence of NSP1 on the ribosome, viral translation proceeds however. The molecular mechanism of the so-called viral evasion to NSP1 inhibition remains elusive. Here, we confirm that viral translation is maintained in the presence of NSP1. The evasion to NSP1-inhibition is mediated by the cis-acting RNA hairpin SL1 in the 5'UTR of SARS-CoV-2. NSP1-evasion can be transferred on a reporter transcript by SL1 transplantation. The apical part of SL1 is only required for viral translation. We show that NSP1 remains bound on the ribosome during viral translation. We suggest that the interaction between NSP1 and SL1 frees the mRNA accommodation channel while maintaining NSP1 bound to the ribosome. Thus, NSP1 acts as a ribosome gatekeeper, shutting down host translation or fostering SARS-CoV-2 translation depending on the presence of the SL1 5'UTR hairpin. SL1 is also present and necessary for translation of sub-genomic RNAs in the late phase of the infectious program. Consequently, therapeutic strategies targeting SL1 should affect viral translation at early and late stages of infection. Therefore, SL1 might be seen as a genuine 'Achille heel' of the virus.

The nature of the purine at position 34 in tRNAs of 4-codon boxes is correlated with nucleotides at positions 32 and 38 to maintain decoding fidelity.

Translation fidelity relies essentially on the ability of ribosomes to accurately recognize triplet interactions between codons on mRNAs and anticodons of tRNAs. To determine the codon-anticodon pairs that are efficiently accepted by the eukaryotic ribosome, we took advantage of the IRES from the intergenic region (IGR) of the Cricket Paralysis Virus. It contains an essential pseudoknot PKI that structurally and functionally mimics a codon-anticodon helix. We screened the entire set of 4096 possible combinations using ultrahigh-throughput screenings combining coupled transcription/translation and droplet-based microfluidics. Only 97 combinations are efficiently accepted and accommodated for translocation and further elongation: 38 combinations involve cognate recognition with Watson-Crick pairs and 59 involve near-cognate recognition pairs with at least one mismatch. More than half of the near-cognate combinations (36/59) contain a G at the first position of the anticodon (numbered 34 of tRNA). G34-containing tRNAs decoding 4-codon boxes are almost absent from eukaryotic genomes in contrast to bacterial genomes. We reconstructed these missing tRNAs and could demonstrate that these tRNAs are toxic to cells due to their miscoding capacity in eukaryotic translation systems. We also show that the nature of the purine at position 34 is correlated with the nucleotides present at 32 and 38.

A tRNA-mimic Strategy to Explore the Role of G34 of tRNAGly in Translation and Codon Frameshifting.

Decoding of the 61 sense codons of the genetic code requires a variable number of tRNAs that establish codon-anticodon interactions. Thanks to the wobble base pairing at the third codon position, less than 61 different tRNA isoacceptors are needed to decode the whole set of codons. On the tRNA, a subtle distribution of nucleoside modifications shapes the anticodon loop structure and participates to accurate decoding and reading frame maintenance. Interestingly, although the 61 anticodons should exist in tRNAs, a strict absence of some tRNAs decoders is found in several codon families. For instance, in Eukaryotes, G34-containing tRNAs translating 3-, 4- and 6-codon boxes are absent. This includes tRNA specific for Ala, Arg, Ile, Leu, Pro, Ser, Thr, and Val. tRNAGly is the only exception for which in the three kingdoms, a G34-containing tRNA exists to decode C3 and U3-ending codons. To understand why G34-tRNAGly exists, we analysed at the genome wide level the codon distribution in codon +1 relative to the four GGN Gly codons. When considering codon GGU, a bias was found towards an unusual high usage of codons starting with a G whatever the amino acid at +1 codon. It is expected that GGU codons are decoded by G34-containing tRNAGly, decoding also GGC codons. Translation studies revealed that the presence of a G at the first position of the downstream codon reduces the +1 frameshift by stabilizing the G34•U3 wobble interaction. This result partially explains why G34-containing tRNAGly exists in Eukaryotes whereas all the other G34-containing tRNAs for multiple codon boxes are absent.

START: STructure-Assisted RNA Translation.

Cap-dependent translation initiation begins by assembly of a pre-initiation ribosomal complex that scans the 5' Untranslated Region in order to localise the start codon. During this process, RNA secondary structures are melted by RNA helicases. Guenther et al reported that the yeast helicase Ded1, an orthologue of the mammalian DDX3 helicase, is responsible for this activity. When Ded1 is non-functional, RNA structures in the 5'UTR promote translation initiation on Alternative Translation Initiation Sites (ATIS) lead to uORF translation and consequently down-regulation of the main ORF. This mechanism is driven by the sole presence of RNA secondary structures located downstream of ATIS. Translation initiation mediated by RNA structures is found in other messenger RNAs. We propose to name this novel mechanism STructure-Assisted-RNA-Translation or START.

Tracking the m7G-cap during translation initiation by crosslinking methods.

In eukaryotes, cap-dependent translation initiation is a sophisticated process that requires numerous trans-acting factors, the eukaryotic Initiation Factors (eIFs). Their main function is to assist the ribosome for accurate AUG start codon recognition. The whole process requires a 5'-3' scanning step and is therefore highly dynamic. Therefore translation requires a complex interplay between eIFs through assembly/release cycles. Here, we describe an original approach to assess the dynamic features of translation initiation. The principle is to use the m7Gcap located at the 5' extremity of mRNAs as a tracker to monitor RNA and protein components that are in its vicinity. Cap-binding molecules are trapped by chemical and UV crosslinking. The combination of cap crosslinking methods in cell-free translation systems with the use of specific translation inhibitors for different steps such as edeine, GMP-PNP or cycloheximide allowed assessing the cap fate during eukaryotic translation. Here, we followed the position of the cap in the histone H4 mRNA and the cap binding proteins during H4 mRNA translation.