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.

The interplay between cis- and trans-acting factors drives selective mRNA translation initiation in eukaryotes.

Translation initiation consists in the assembly of the small and large ribosomal subunits on the start codon. This important step directly modulates the general proteome in living cells. Recently, genome wide studies revealed unexpected translation initiation events from unsuspected novel open reading frames resulting in the synthesis of a so-called 'dark proteome'. Indeed, the identification of the start codon by the translation machinery is a critical step that defines the translational landscape of the cell. Therefore, translation initiation is a highly regulated process in all organisms. In this review, we focus on the various cis- and trans-acting factors that rule the regulation of translation initiation in eukaryotes. Recent discoveries have shown that the guidance of the translation machinery for the choice of the start codon require sophisticated molecular mechanisms. In particular, the 5'UTR and the coding sequences contain cis-acting elements that trigger the use of AUG codons but also non-AUG codons to initiate protein synthesis. The use of these alternative start codons is also largely influenced by numerous trans-acting elements that drive selective mRNA translation in response to environmental changes.

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 Toll pathway mediates Drosophila resilience to Aspergillus mycotoxins through specific Bomanins.

Host defense against infections encompasses both resistance, which targets microorganisms for neutralization or elimination, and resilience/disease tolerance, which allows the host to withstand/tolerate pathogens and repair damages. In Drosophila, the Toll signaling pathway is thought to mediate resistance against fungal infections by regulating the secretion of antimicrobial peptides, potentially including Bomanins. We find that Aspergillus fumigatus kills Drosophila Toll pathway mutants without invasion because its dissemination is blocked by melanization, suggesting a role for Toll in host defense distinct from resistance. We report that mutants affecting the Toll pathway or the 55C Bomanin locus are susceptible to the injection of two Aspergillus mycotoxins, restrictocin and verruculogen. The vulnerability of 55C deletion mutants to these mycotoxins is rescued by the overexpression of Bomanins specific to each challenge. Mechanistically, flies in which BomS6 is expressed in the nervous system exhibit an enhanced recovery from the tremors induced by injected verruculogen and display improved survival. Thus, innate immunity also protects the host against the action of microbial toxins through secreted peptides and thereby increases its resilience to infection.

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.

Tau mRNA Metabolism in Neurodegenerative Diseases: A Tangle Journey.

Tau proteins are known to be mainly involved in regulation of microtubule dynamics. Besides this function, which is critical for axonal transport and signal transduction, tau proteins also have other roles in neurons. Moreover, tau proteins are turned into aggregates and consequently trigger many neurodegenerative diseases termed tauopathies, of which Alzheimer's disease (AD) is the figurehead. Such pathological aggregation processes are critical for the onset of these diseases. Among the various causes of tau protein pathogenicity, abnormal tau mRNA metabolism, expression and dysregulation of tau post-translational modifications are critical steps. Moreover, the relevance of tau function to general mRNA metabolism has been highlighted recently in tauopathies. In this review, we mainly focus on how mRNA metabolism impacts the onset and development of tauopathies. Thus, we intend to portray how mRNA metabolism of, or mediated by, tau is associated with neurodegenerative diseases.

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.

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.

How Many Messenger RNAs Can Be Translated by the START Mechanism?

Translation initiation is a key step in the protein synthesis stage of the gene expression pathway of all living cells. In this important process, ribosomes have to accurately find the AUG start codon in order to ensure the integrity of the proteome. "Structure Assisted RNA Translation", or "START", has been proposed to use stable secondary structures located in the coding sequence to augment start site selection by steric hindrance of the progression of pre-initiation complex on messenger RNA. This implies that such structures have to be located downstream and at on optimal distance from the AUG start codon (i.e., downstream nucleotide +16). In order to assess the importance of the START mechanism in the overall mRNA translation process, we developed a bioinformatic tool to screen coding sequences for such stable structures in a 50 nucleotide-long window spanning the nucleotides from +16 to +65. We screened eight bacterial genomes and six eukaryotic genomes. We found stable structures in 0.6-2.5% of eukaryotic coding sequences. Among these, approximately half of them were structures predicted to form G-quadruplex structures. In humans, we selected 747 structures. In bacteria, the coding sequences from Gram-positive bacteria contained 2.6-4.2% stable structures, whereas the structures were less abundant in Gram-negative bacteria (0.2-2.7%). In contrast to eukaryotes, putative G-quadruplex structures are very rare in the coding sequence of bacteria. Altogether, our study reveals that the START mechanism seems to be an ancient strategy to facilitate the start codon recognition that is used in different kingdoms of life.

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.

Identification of 6-amino-1H-pyrazolo[3,4-d]pyrimidines with in vivo efficacy against visceral leishmaniasis.

Visceral leishmaniasis (VL) affects millions of people across the world, largely in developing nations. It is fatal if left untreated and the current treatments are inadequate. As such, there is an urgent need for new, improved medicines. In this paper, we describe the identification of a 6-amino-N-(piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidine scaffold and its optimization to give compounds which showed efficacy when orally dosed in a mouse model of VL.

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.

A variant in MRPS14 (uS14m) causes perinatal hypertrophic cardiomyopathy with neonatal lactic acidosis, growth retardation, dysmorphic features and neurological involvement.

Dysfunction of mitochondrial translation is an increasingly important molecular cause of human disease, but structural defects of mitochondrial ribosomal subunits are rare. We used next-generation sequencing to identify a homozygous variant in the mitochondrial small ribosomal protein 14 (MRPS14, uS14m) in a patient manifesting with perinatal hypertrophic cardiomyopathy, growth retardation, muscle hypotonia, elevated lactate, dysmorphy and mental retardation. In skeletal muscle and fibroblasts from the patient, there was biochemical deficiency in complex IV of the respiratory chain. In fibroblasts, mitochondrial translation was impaired, and ectopic expression of a wild-type MRPS14 cDNA functionally complemented this defect. Surprisingly, the mutant uS14m was stable and did not affect assembly of the small ribosomal subunit. Instead, structural modeling of the uS14m mutation predicted a disruption to the ribosomal mRNA channel.Collectively, our data demonstrate pathogenic mutations in MRPS14 can manifest as a perinatal-onset mitochondrial hypertrophic cardiomyopathy with a novel molecular pathogenic mechanism that impairs the function of mitochondrial ribosomes during translation elongation or mitochondrial mRNA recruitment rather than assembly.

Identification of GSK3186899/DDD853651 as a Preclinical Development Candidate for the Treatment of Visceral Leishmaniasis.

The leishmaniases are diseases that affect millions of people across the world, in particular visceral leishmaniasis (VL) which is fatal unless treated. Current standard of care for VL suffers from multiple issues and there is a limited pipeline of new candidate drugs. As such, there is a clear unmet medical need to identify new treatments. This paper describes the optimization of a phenotypic hit against Leishmania donovani, the major causative organism of VL. The key challenges were to balance solubility and metabolic stability while maintaining potency. Herein, strategies to address these shortcomings and enhance efficacy are discussed, culminating in the discovery of preclinical development candidate GSK3186899/DDD853651 (1) for VL.