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.

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.

CUG initiation and frameshifting enable production of dipeptide repeat proteins from ALS/FTD C9ORF72 transcripts.

Expansion of G4C2 repeats in the C9ORF72 gene is the most prevalent inherited form of amyotrophic lateral sclerosis and frontotemporal dementia. Expanded transcripts undergo repeat-associated non-AUG (RAN) translation producing dipeptide repeat proteins from all reading frames. We determined cis-factors and trans-factors influencing translation of the human C9ORF72 transcripts. G4C2 translation operates through a 5'-3' cap-dependent scanning mechanism, requiring a CUG codon located upstream of the repeats and an initiator Met-tRNAMeti. Production of poly-GA, poly-GP, and poly-GR proteins from the three frames is influenced by mutation of the same CUG start codon supporting a frameshifting mechanism. RAN translation is also regulated by an upstream open reading frame (uORF) present in mis-spliced C9ORF72 transcripts. Inhibitors of the pre-initiation ribosomal complex and RNA antisense oligonucleotides selectively targeting the 5'-flanking G4C2 sequence block ribosomal scanning and prevent translation. Finally, we identified an unexpected affinity of expanded transcripts for the ribosomal subunits independently from translation.

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.

The IRES5'UTR of the dicistrovirus cricket paralysis virus is a type III IRES containing an essential pseudoknot structure.

Cricket paralysis virus (CrPV) is a dicistrovirus. Its positive-sense single-stranded RNA genome contains two internal ribosomal entry sites (IRESs). The 5' untranslated region (5'UTR) IRES5'UTR mediates translation of non-structural proteins encoded by ORF1 whereas the well-known intergenic region (IGR) IRESIGR is required for translation of structural proteins from open reading frame 2 in the late phase of infection. Concerted action of both IRES is essential for host translation shut-off and viral translation. IRESIGR has been extensively studied, in contrast the IRES5'UTR remains largely unexplored. Here, we define the minimal IRES element required for efficient translation initiation in drosophila S2 cell-free extracts. We show that IRES5'UTR promotes direct recruitment of the ribosome on the cognate viral AUG start codon without any scanning step, using a Hepatitis-C virus-related translation initiation mechanism. Mass spectrometry analysis revealed that IRES5'UTR recruits eukaryotic initiation factor 3, confirming that it belongs to type III class of IRES elements. Using Selective 2'-hydroxyl acylation analyzed by primer extension and DMS probing, we established a secondary structure model of 5'UTR and of the minimal IRES5'UTR. The IRES5'UTR contains a pseudoknot structure that is essential for proper folding and ribosome recruitment. Overall, our results pave the way for studies addressing the synergy and interplay between the two IRES from CrPV.

Purification of mRNA-programmed translation initiation complexes suitable for mass spectrometry analysis.

Liquid Chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS) is a powerful analytical technique for the identification and mass analysis of complex protein mixtures. Here, we present a combination of methods developed for the extensive/deep proteomic analysis of purified ribosome/mRNA particles assembled in rabbit reticulocyte lysate (RRL). Ribosomes are assembled on chimeric biotinylated mRNA-DNA molecules immobilized on streptavidin-coated beads and incubated with RRL to form initiation complexes. After washing steps, the complexes are trypsin-digested directly on the beads in semi-native condition or after their elution from the beads in denaturing Laemmli buffer. The nanoLC-MS/MS analysis performed on complexes assembled on ß-globin, viral HCV, and histone H4 mRNAs revealed significant differences in initiation factors composition in agreement with models of translation initiation used by these different types of mRNAs. Using Laemmli-denaturing condition induces release of deeply buried peptides from the ribosome and eukaryotic initiation factor 3 (eIF3) allowing the identification of the nearly complete set of ribosomal proteins.

Rapid purification of ribosomal particles assembled on histone H4 mRNA: a new method based on mRNA-DNA chimaeras.

Detailed knowledge of the structure of the ribosomal particles during their assembly on mRNA is a prerequisite for understanding the intricate translation initiation process. In vitro preparation of eukaryotic translation initiation complexes is limited by the rather tricky assembly from individually purified ribosomal subunits, initiation factors and initiator tRNA. In order to directly isolate functional complexes from living cells, methods based on affinity tags have been developed which, however, often suffer from non-specific binding of proteins and/or RNAs. In the present study we present a novel method designed for the purification of high-quality ribosome/mRNA particles assembled in RRL (rabbit reticulocyte lysate). Chimaerical mRNA-DNA molecules, consisting of the full-length mRNA ligated to a biotinylated desoxy-oligonucleotide, are immobilized on streptavidin-coated beads and incubated with RRL to form initiation complexes. After a washing step, the complexes are eluted by specific DNase I digestion of the DNA moiety of the chimaera, releasing initiation complexes in native conditions. Using this simple and robust purification setup, 80S particles properly programmed with full-length histone H4 mRNA were isolated with the expected ribosome/mRNA molar ratio of close to 1. We show that by using this novel approach purified ribosomal particles can be obtained that are suitable for biochemical and structural studies, in particular single-particle cryo-EM (cryo-electron microscopy). This purification method thus is a versatile tool for the isolation of fully functional RNA-binding proteins and macromolecular RNPs.

Cap-assisted internal initiation of translation of histone H4.

In eukaryotes, a crucial step of translation initiation is the binding of the multifactor complex eIF4F to the 5' end of the mRNA, a prerequisite to recruitment of the activated small ribosomal 43S particle. Histone H4 mRNAs have short 5'UTRs, which do not conform to the conventional scanning-initiation model. Here we show that the ORF of histone mRNA contains two structural elements critical for translation initiation. One of the two structures binds eIF4E without the need of the cap. Ribosomal 43S particles become tethered to this site and directly loaded in the vicinity of the AUG. The other structure, 19 nucleotides downstream of the initiation codon, forms a three-way helix junction, which sequesters the m(7)G cap. This element facilitates direct positioning of the ribosome on the cognate start codon. This unusual translation initiation mode might be considered as a hybrid mechanism between the canonical and the IRES-driven translation initiation process.