Gene expression is a multistep process that involves the transcription, translation and turnover of messenger RNAs and proteins. Although it is one of the most fundamental processes of life, the entire cascade has never been quantified on a genome-wide scale. Here we simultaneously measured absolute mRNA and protein abundance and turnover by parallel metabolic pulse labelling for more than 5,000 genes in mammalian cells. Whereas mRNA and protein levels correlated better than previously thought, corresponding half-lives showed no correlation. Using a quantitative model we have obtained the first genome-scale prediction of synthesis rates of mRNAs and proteins. We find that the cellular abundance of proteins is predominantly controlled at the level of translation. Genes with similar combinations of mRNA and protein stability shared functional properties, indicating that half-lives evolved under energetic and dynamic constraints. Quantitative information about all stages of gene expression provides a rich resource and helps to provide a greater understanding of the underlying design principles.

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Global quantification of mammalian gene expression control

Techniques for systematically monitoring protein translation have lagged far behind methods for measuring messenger RNA (mRNA) levels. Here, we present a ribosome-profiling strategy that is based on the deep sequencing of ribosome-protected mRNA fragments and enables genome-wide investigation of translation with subcodon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control in both determining absolute protein abundance and responding to environmental stress. We also observed distinct phases during translation that involve a large decrease in ribosome density going from early to late peptide elongation as well as widespread regulated initiation at non-adenine-uracil-guanine (AUG) codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible.

/pdf/genome-wide-analysis-in-vivo-of-translation-with-nucleotide-36vshepmua.pdf

Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling

Despite their name, synonymous mutations have significant consequences for cellular processes in all taxa. As a result, an understanding of codon bias is central to fields as diverse as molecular evolution and biotechnology. Although recent advances in sequencing and synthetic biology have helped to resolve longstanding questions about codon bias, they have also uncovered striking patterns that suggest new hypotheses about protein synthesis. Ongoing work to quantify the dynamics of initiation and elongation is as important for understanding natural synonymous variation as it is for designing transgenes in applied contexts.

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Synonymous but not the same: the causes and consequences of codon bias.

Cellular states are determined by differential expression of the cell’s proteins. The relationship between protein and mRNA expression levels informs about the combined outcomes of translation and protein degradation which are, in addition to transcription and mRNA stability, essential contributors to gene expression regulation. This review summarizes the state of knowledge about large-scale measurements of absolute protein and mRNA expression levels, and the degree of correlation between the two parameters. We summarize the information that can be derived from comparison of protein and mRNA expression levels and discuss how corresponding sequence characteristics suggest modes of regulation.

/pdf/global-signatures-of-protein-and-mrna-expression-levels-1qve1qwtri.pdf

Global signatures of protein and mRNA expression levels

Advances over the past few years have begun to enable prediction and design of macromolecular structures at near-atomic accuracy. Progress has stemmed from the development of reasonably accurate and efficiently computed all-atom potential functions as well as effective conformational sampling strategies appropriate for searching a highly rugged energy landscape, both driven by feedback from structure prediction and design tests. A unified energetic and kinematic framework in the Rosetta program allows a wide range of molecular modeling problems, from fibril structure prediction to RNA folding to the design of new protein interfaces, to be readily investigated and highlights areas for improvement. The methodology enables the creation of novel molecules with useful functions and holds promise for accelerating experimental structural inference. Emerging connections to crystallographic phasing, NMR modeling, and lower-resolution approaches are described and critically assessed.

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Macromolecular Modeling with Rosetta

Artifacts are commonly encountered in clinical CT and may obscure or simulate pathology. There are many different types of CT artifacts, including noise, beam hardening, scatter, pseudoenhancement, motion, cone-beam, helical, ring and metal artifacts. We review the cause and appearance of each type of artifact, correct some popular misconceptions and describe modern techniques for artifact reduction. Noise can be reduced using iterative reconstruction or by combining data from multiple scans. This enables lower radiation dose and higher resolution scans. Metal artifacts can also be reduced using iterative reconstruction, resulting in a more accurate diagnosis. Dual- and multi-energy (photon counting) CT can reduce beam hardening and provide better tissue contrast. Methods for reducing noise and out-of-field artifacts may enable ultra-high resolution limited field of view imaging of tumors and other structures.

/pdf/ct-artifacts-causes-and-reduction-techniques-3km19o9ami.pdf

CT artifacts: Causes and reduction techniques

Multidetector CT facilitates reduced metal artifacts in images from simulated and clinical scans and has the potential to improve diagnostic accuracy.

Evaluation of Two Iterative Techniques for Reducing Metal Artifacts in Computed Tomography

Several recently introduced ‘new’ techniques in computed tomography—iterative reconstruction, gated cardiac CT, multiple-source, and dual-energy CT—actually date back to the early days of CT. We review the historic origins and evolution of these techniques, which may provide some insight into the latest innovations in commercial CT systems.

/pdf/computed-tomography-old-ideas-and-new-technology-3ckzuclq8f.pdf

Computed tomography—old ideas and new technology

Potential energy functions for protein design.

Translation of an mRNA is generally divided into three stages: initiation, elongation and termination. The relative rates of these steps determine both the number and position of ribosomes along the mRNA, but traditional velocity sedimentation assays for the translational status of mRNA determine only the number of bound ribosomes. We developed a procedure, termed Ribosome Density Mapping (RDM), that uses sitespecific cleavage of polysomal mRNA followed by separation on a sucrose gradient and northern analysis, to determine the number of ribosomes associated with specified portions of a particular mRNA. This procedure allows us to test models for translation and its control, and to examine properties of individual steps of translation in vivo. We tested specific predictions from the current model for translational control of GCN4 expression in yeast and found that ribosomes were differentially associated with the uORFs elements and coding region under different growth conditions, consistent with this model. We also mapped ribosome density along the ORF of several mRNAs, to probe basic kinetic properties of translational steps in yeast. We found no detectable decline in ribosome density between the 5 0 and 3 0 ends of the ORFs, suggesting that the average processivity of elongation is very high. Conversely, there was no queue of ribosomes at the termination site, suggesting that termination is not very slow relative to elongation and initiation. Finally, the RDM results suggest that less frequent initiation of translation on mRNAs with longer ORFs is responsible for the inverse correlation between ORF length and ribosomal density that we observed in a global analysis of translation. These results provide new insights into eukaryotic translation in vivo.

/pdf/dissecting-eukaryotic-translation-and-its-control-by-2cwmsstcsh.pdf

F. Edward Boas

Papers

CT artifacts: Causes and reduction techniques

Evaluation of Two Iterative Techniques for Reducing Metal Artifacts in Computed Tomography

Computed tomography—old ideas and new technology

Potential energy functions for protein design.

Dissecting eukaryotic translation and its control by ribosome density mapping