Quantitative principles of cis-translational control by general mRNA sequence features in eukaryotes.

BackgroundGeneral translational cis-elements are present in the mRNAs of all genes and affect the recruitment, assembly, and progress of preinitiation complexes and the ribosome under many physiological states. These elements include mRNA folding, upstream open reading frames, specific nucleotides flanking the initiating AUG codon, protein coding sequence length, and codon usage. The quantitative contributions of these sequence features and how and why they coordinate to control translation rates are not well understood.ResultsHere, we show that these sequence features specify 42-81% of the variance in translation rates in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Mus musculus, and Homo sapiens. We establish that control by RNA secondary structure is chiefly mediated by highly folded 25-60 nucleotide segments within mRNA 5' regions, that changes in tri-nucleotide frequencies between highly and poorly translated 5' regions are correlated between all species, and that control by distinct biochemical processes is extensively correlated as is regulation by a single process acting in different parts of the same mRNA.ConclusionsOur work shows that general features control a much larger fraction of the variance in translation rates than previously realized. We provide a more detailed and accurate understanding of the aspects of RNA structure that directs translation in diverse eukaryotes. In addition, we note that the strongly correlated regulation between and within cis-control features will cause more even densities of translational complexes along each mRNA and therefore more efficient use of the translation machinery by the cell

A Hybrid of Metabolic Flux Analysis and Bayesian Factor Modeling for Multiomic Temporal Pathway Activation.

The growing availability of multiomic data provides a highly comprehensive view of cellular processes at the levels of mRNA, proteins, metabolites, and reaction fluxes. However, due to probabilistic interactions between components depending on the environment and on the time course, casual, sometimes rare interactions may cause important effects in the cellular physiology. To date, interactions at the pathway level cannot be measured directly, and methodologies to predict pathway cross-correlations from reaction fluxes are still missing. Here, we develop a multiomic approach of flux-balance analysis combined with Bayesian factor modeling with the aim of detecting pathway cross-correlations and predicting metabolic pathway activation profiles. Starting from gene expression profiles measured in various environmental conditions, we associate a flux rate profile with each condition. We then infer pathway cross-correlations and identify the degrees of pathway activation with respect to the conditions and time course using Bayesian factor modeling. We test our framework on the most recent metabolic reconstruction of Escherichia coli in both static and dynamic environments, thus predicting the functionality of particular groups of reactions and how it varies over time. In a dynamic environment, our method can be readily used to characterize the temporal progression of pathway activation in response to given stimuli

mTORC1-mediated translational elongation limits intestinal tumour initiation and growth.

Inactivation of APC is a strongly predisposing event in the development of colorectal cancer, prompting the search for vulnerabilities specific to cells that have lost APC function. Signalling through the mTOR pathway is known to be required for epithelial cell proliferation and tumour growth, and the current paradigm suggests that a critical function of mTOR activity is to upregulate translational initiation through phosphorylation of 4EBP1 (refs 6, 7). This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 (ref. 8), would be ineffective in limiting cancer progression in APC-deficient lesions. Here we show in mice that mTOR complex 1 (mTORC1) activity is absolutely required for the proliferation of Apc-deficient (but not wild-type) enterocytes, revealing an unexpected opportunity for therapeutic intervention. Although APC-deficient cells show the expected increases in protein synthesis, our study reveals that it is translation elongation, and not initiation, which is the rate-limiting component. Mechanistically, mTORC1-mediated inhibition of eEF2 kinase is required for the proliferation of APC-deficient cells. Importantly, treatment of established APC-deficient adenomas with rapamycin (which can target eEF2 through the mTORC1-S6K-eEF2K axis) causes tumour cells to undergo growth arrest and differentiation. Taken together, our data suggest that inhibition of translation elongation using existing, clinically approved drugs, such as the rapalogs, would provide clear therapeutic benefit for patients at high risk of developing colorectal cancer

DNA intercalation without flipping in the specific ThaI DNA complex

The PD-(D/E)XK type II restriction endonuclease ThaI cuts the target sequence CG/CG with blunt ends. Here, we report the 1.3 Å resolution structure of the enzyme in complex with substrate DNA and a sodium or calcium ion taking the place of a catalytic magnesium ion. The structure identifies Glu54, Asp82 and Lys93 as the active site residues. This agrees with earlier bioinformatic predictions and implies that the PD and (D/E)XK motifs in the sequence are incidental. DNA recognition is very unusual: the two Met47 residues of the ThaI dimer intercalate symmetrically into the CG steps of the target sequence. They approach the DNA from the minor groove side and penetrate the base stack entirely. The DNA accommodates the intercalating residues without nucleotide flipping by a doubling of the CG step rise to twice its usual value, which is accompanied by drastic unwinding. Displacement of the Met47 side chains from the base pair midlines toward the downstream CG steps leads to large and compensating tilts of the first and second CG steps. DNA intercalation by ThaI is unlike intercalation by HincII, HinP1I or proteins that bend or repair DNA

The Leishmania PABP1–eIF4E4 interface: a novel 5′–3′ interaction architecture for trans-spliced mRNAs

Trans-splicing of trypanosomatid polycistronic transcripts produces polyadenylated monocistronic mRNAs modified to form the 5′ cap4 structure (m7Gpppm36,6,2′Apm2′Apm2′Cpm23,2′U). NMR and X-ray crystallography reveal that Leishmania has a unique type of N-terminally-extended cap-binding protein (eIF4E4) that binds via a PAM2 motif to PABP1. This relies on the interactions of a combination of polar and charged amino acid side-chains together with multiple hydrophobic interactions, and underpins a novel architecture in the Leishmania cap4-binding translation factor complex. Measurements using microscale thermophoresis, fluorescence anisotropy and surface plasmon resonance characterize the key interactions driving assembly of the Leishmania translation initiation complex. We demonstrate that this complex can accommodate Leishmania eIF4G3 which, unlike the standard eukaryotic initiation complex paradigm, binds tightly to eIF4E4, but not to PABP1. Thus, in Leishmania, the chain of interactions 5′cap4-eIF4E4–PABP1-poly(A) bridges the mRNA 5′ and 3′ ends. Exceptionally, therefore, by binding tightly to two protein ligands and to the mRNA 5′ cap4 structure, the trypanosomatid N-terminally extended form of eIF4E acts as the core molecular scaffold for the mRNA-cap-binding complex. Finally, the eIF4E4 N-terminal extension is an intrinsically disordered region that transitions to a partly folded form upon binding to PABP1, whereby this interaction is not modulated by poly(A) binding to PABP

Translation Initiation Factors eIF3 and HCR1 Control Translation Termination and Stop Codon Read-Through in Yeast Cells

Translation is divided into initiation, elongation, termination and ribosome recycling. Earlier work implicated several eukaryotic initiation factors (eIFs) in ribosomal recycling in vitro. Here, we uncover roles for HCR1 and eIF3 in translation termination in vivo. A substantial proportion of eIF3, HCR1 and eukaryotic release factor 3 (eRF3) but not eIF5 (a well-defined “initiation-specific” binding partner of eIF3) specifically co-sediments with 80S couples isolated from RNase-treated heavy polysomes in an eRF1-dependent manner, indicating the presence of eIF3 and HCR1 on terminating ribosomes. eIF3 and HCR1 also occur in ribosome- and RNA-free complexes with both eRFs and the recycling factor ABCE1/RLI1. Several eIF3 mutations reduce rates of stop codon read-through and genetically interact with mutant eRFs. In contrast, a slow growing deletion of hcr1 increases read-through and accumulates eRF3 in heavy polysomes in a manner suppressible by overexpressed ABCE1/RLI1. Based on these and other findings we propose that upon stop codon recognition, HCR1 promotes eRF3·GDP ejection from the post-termination complexes to allow binding of its interacting partner ABCE1/RLI1. Furthermore, the fact that high dosage of ABCE1/RLI1 fully suppresses the slow growth phenotype of hcr1? as well as its termination but not initiation defects implies that the termination function of HCR1 is more critical for optimal proliferation than its function in translation initiation. Based on these and other observations we suggest that the assignment of HCR1 as a bona fide eIF3 subunit should be reconsidered. Together our work characterizes novel roles of eIF3 and HCR1 in stop codon recognition, defining a communication bridge between the initiation and termination/recycling phases of translation