Stable m7G Cap-Distal 5'UTR Hairpin Structure Mediates Distinct 40S and 60S Binding Dynamics

The 5' untranslated region (UTR) of diverse mRNAs contains secondary structures that can influence protein synthesis by modulating the initiation step of translation. Studies support the ability of these structures to inhibit 40S subunit recruitment and scanning, but the dynamics of ribosomal subunit interactions with mRNA remain poorly understood. Here, we developed a reconstituted Saccharomyces cerevisiae cell-free translation system with fluorescently labeled ribosomal subunits. We applied this extract and single-molecule fluorescence microscopy to monitor, in real time, individual 40S and 60S interactions with mRNAs containing 5' UTR hairpin structures with varying thermostability. In comparison to mRNAs containing no or weak 5' UTR hairpins (ΔG >= -5.4 kcal/mol), mRNAs with stable hairpins (ΔG <= -16.5 kcal/mol) showed reduced numbers of 60S recruitment to mRNA, consistent with the expectation of reduced translation efficiency for such mRNAs. Interestingly, such mRNAs showed increased numbers of 40S recruitment events to individual mRNAs but with shortened duration on mRNA. Correlation analysis showed that these unstable 40S binding events were nonproductive for 60S recruitment. Furthermore, although the mRNA sequence is long enough to accommodate multiple 40S, individual mRNAs are predominantly observed to engage with a single 40S at a time, indicating the sequestering of mRNA 5' end by initiating 40S. Altogether, these observations suggest that stable cap-distal hairpins in 5' UTR reduce initiation and translation efficiency by destabilizing 40S-mRNA interactions and promoting 40S dissociation from mRNA. The premature 40S dissociation frees mRNA 5'-end accessibility for new initiation events, but the increased rate of 40S recruitment is insufficient to compensate for the reduction of initiation efficiency due to premature 40S dissociation. This study provides the first single-molecule kinetic characterization of 40S/60S interactions with mRNA during cap-dependent initiation and the modulation of such interactions by cap-distal 5' UTR hairpin structures.

Progressive alterations of striatal polysomal architecture in a mouse model of Huntington's disease

Neurons rely on a precise spatial and temporal control of protein synthesis due to their highly polarized morphology and their functional singularities. Consequently, alterations in protein translation have been widely related to the development and progression of various neurological and neurodegenerative disorders, including Huntington's disease. Here we explored the architecture of polysomes in their native brain context by performing 3D electron tomography of striatal tissue derived from a knock-in mouse model of the disease. Results showed a progressive remodelling towards a polysomal compacted architecture that parallels in time the emergence and progression of symptoms in the mouse model. The aberrant architecture is compatible with ribosome stalling phenomena and, in fact, we detected an increase in the expression of the stalling release factor eIF5A2. Polysomal sedimentation gradients showed significant excess in the accumulation of free 40S ribosomal subunits in heterozygous striatal samples. Overall the results indicate that changes in the architecture of the protein synthesis machinery might be at the basis of translational alterations associated to Huntington's disease and open new avenues for understanding disease progression.

Comparative and phylogenomic analyses of mitochondrial genomes in Coccinellidae (Coleoptera: Coccinelloidea)

The Coccinellidae are one of the most familiar beetle families, the ladybirds. Despite the great ecological and economic significance, the phylogenetic relationships of Coccinellidae remain poorly understood. One of the reasons is that the sequenced mitogenomes available for this family are very limited. We sequenced complete or nearly complete mitogenomes from seven species of the tribe Coccinellini with next-generation sequencing. All species have the same gene content and gene order as the putatively ancestral insect mitogenome. A large intergenic spacer region (> 890 bp) was found located between trnI and trnQ. The potential for using secondary structures of the large and small ribosomal subunits for phylogenetic reconstruction was predicted. The phylogenetic relationships were explored through comparative analyses across more than 30 coccinellid species. We performed phylogenetic analyses with both concatenation methods (Maximum Likelihood and Bayesian Inference) and multispecies coalescent method (ASTRAL). Phylogenetic results strongly supported the monophyly of Coccinellidae. Within Coccinellidae, the Epilachnini and the Coccinellini including Halyziini were monophyletic, while the Scymnini and Coccidulini were non-monophyletic.

eIF5B and eIF1A remodel human translation initiation complexes to mediate ribosomal subunit joining

Joining of the ribosomal subunits at a translation start site on a messenger RNA during initiation commits the ribosome to synthesize a protein. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when universally-conserved eukaryotic initiation factors (eIFs) eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we examined initiation complexes that contained both eIF1A and eIF5B using single-particle electron cryo-microscopy. The resulting structure illuminated how eukaryote-specific contacts between eIF1A and eIF5B remodel the initiation complex to orient initiator tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during human translation initiation.

Systematic mapping of rRNA 2'-O methylation during frog development and involvement of the methyltransferase Fibrillarin in eye and craniofacial development in Xenopus laevis

Ribosomes are essential nanomachines responsible for protein production. Although ribosomes are present in every living cell, ribosome biogenesis dysfunction diseases, called ribosomopathies, impact particular tissues specifically. Here, we evaluate the importance of the box C/D snoRNA-associated ribosomal RNA methyltransferase fibrillarin (Fbl) in the early embryonic development of Xenopus laevis. We report that in developing embryos, the neural plate, neural crest cells (NCCs), and NCC derivatives are rich in fbl transcripts. Fbl knockdown leads to striking morphological defects affecting the eyes and craniofacial skeleton, due to lack of NCC survival caused by massive p53-dependent apoptosis. Fbl is required for efficient pre-rRNA processing and 18S rRNA production, which explains the early developmental defects. Using RiboMethSeq, we systematically reinvestigated ribosomal RNA 2'-O methylation in X. laevis, confirming all 89 previously mapped sites and identifying 15 novel putative positions in 18S and 28S rRNA. Twenty-three positions, including 10 of the new ones, were validated orthogonally by low dNTP primer extension. Bioinformatic screening of the X. laevis transcriptome revealed candidate box C/D snoRNAs for all methylated positions. Mapping of 2'-O methylation at six developmental stages in individual embryos indicated a trend towards reduced methylation at specific positions during development. We conclude that fibrillarin knockdown in early Xenopus embryos causes reduced production of functional ribosomal subunits, thus impairing NCC formation and migration.

Macrophage deletion of Noc4l triggers endosomal TLR4/TRIF signal and leads to insulin resistance

In obesity, macrophages drive a low-grade systemic inflammation (LSI) and insulin resistance (IR). The ribosome biosynthesis protein NOC4 (NOC4) mediates 40 S ribosomal subunits synthesis in yeast. Hereby, we reported an unexpected location and function of NOC4L, which was preferentially expressed in human and mouse macrophages. NOC4L was decreased in both obese human and mice. The macrophage-specific deletion of Noc4l in mice displayed IR and LSI. Conversely, Noc4l overexpression by lentivirus treatment and transgenic mouse model improved glucose metabolism in mice. Importantly, we found that Noc4l can interact with TLR4 to inhibit its endocytosis and block the TRIF pathway, thereafter ameliorated LSI and IR in mice.

Structural mechanism of GTPase-powered ribosome-tRNA movement

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP–Pi form stabilizes the rotated conformation of ribosomal subunits and induces twisting of the sarcin-ricin loop of the 23 S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a large-scale rigid-body rotation of EF-G pivoting around the sarcin-ricin loop that facilitates back rotation of the ribosomal subunits and forward swiveling of the head domain of the small subunit, ultimately driving tRNA forward movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.

Ribosomal RNA 2'-O-methylations regulate translation by impacting ribosome dynamics

Protein synthesis by ribosomes is critically important for gene expression in all cells. The ribosomal RNAs (rRNAs) are marked by numerous chemical modifications. An abundant group of rRNA modifications, present in all domains of life, is 2'-O-methylation guided by box C/D small nucleolar RNAs (snoRNAs) which are part of small ribonucleoprotein complexes (snoRNPs). Although 2'-O-methylations are required for proper production of ribosomes, the mechanisms by which these modifications contribute to translation have remained elusive. Here, we show that a change in box C/D snoRNP biogenesis in actively growing yeast cells results in the production of hypo 2'-O-methylated ribosomes with distinct translational properties. Using RiboMeth-Seq for the quantitative analysis of 2'-O methylations, we identify site-specific perturbations of the rRNA 2'-O-methylation pattern and uncover sites that are not required for ribosome production under normal conditions. Characterization of the hypo 2'-O-methylated ribosomes reveals significant translational fidelity defects including frameshifting and near-cognate start codon selection. Using rRNA structural probing, we show that hypo 2'-O-methylation affects the inherent dynamics of the ribosomal subunits and impacts the binding of translation factor eIF1 thereby causing translational defects. Our data reveal an unforeseen spectrum of 2'-O-methylation heterogeneity in yeast rRNA and suggest a significant role for rRNA 2'-O-methylation in regulating cellular translation by controlling ribosome dynamics and ligand binding.

The nucleolar DExD/H protein Hel66 is involved in ribosome biogenesis in Trypanosoma brucei

The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process.

eIF6 rebinding dynamically couples ribosome maturation and translation

Protein synthesis is a cyclical process consisting of translation initiation, elongation, termination and ribosome recycling. The release factors SBDS and EFL1 (both mutated in the leukaemia predisposition disorder Shwachman-Diamond syndrome) license entry of nascent 60S ribosomal subunits into active translation by evicting the anti-association factor eIF6 from the 60S intersubunit face. Here, we show that in mammalian cells, eIF6 holds all free cytoplasmic 60S subunits in a translationally inactive state and that SBDS and EFL1 are the minimal components required to recycle these 60S subunits back into additional rounds of translation by evicting eIF6. Increasing the dose of eIF6 in mice in vivo impairs terminal erythropoiesis by sequestering post-termination 60S subunits in the cytoplasm, disrupting subunit joining and attenuating global protein synthesis. Our data reveal that ribosome maturation and recycling are dynamically coupled by a mechanism that is disrupted in an inherited leukaemia predisposition disorder.