Ribosome Profiling of Mouse Embryonic Stem Cells Reveals the Complexity and Dynamics of Mammalian Proteomes

Most proteins must fold into unique three-dimensional structures to perform their biological functions. In the crowded cellular environment, newly synthesized proteins are at risk of misfolding and forming toxic aggregate species. To ensure efficient folding, different classes of molecular chaperones receive the nascent protein chain emerging from the ribosome and guide it along a productive folding pathway. Because proteins are structurally dynamic, constant surveillance of the proteome by an integrated network of chaperones and protein degradation machineries is required to maintain protein homeostasis (proteostasis). The capacity of this proteostasis network declines during aging, facilitating neurodegeneration and other chronic diseases associated with protein aggregation. Understanding the proteostasis network holds the promise of identifying targets for pharmacological intervention in these pathologies.

/pdf/in-vivo-aspects-of-protein-folding-and-quality-control-s8m996j9sy.pdf

In vivo aspects of protein folding and quality control

Genome-wide analyses of gene expression have so far focused on the abundance of mRNA species as measured either by microarray or, more recently, by RNA sequencing. However, neither approach provides information on protein synthesis, which is the true end point of gene expression. Ribosome profiling is an emerging technique that uses deep sequencing to monitor in vivo translation. Studies using ribosome profiling have already provided new insights into the identity and the amount of proteins that are produced by cells, as well as detailed views into the mechanism of protein synthesis itself.

Ribosome profiling: new views of translation, from single codons to genome scale

https://www.cell.com/molecular-cell/pdf/S1097-2765(15)00402-5.pdf

Codon Bias as a Means to Fine-Tune Gene Expression

The advent of ribosome profiling and other tools to probe mRNA translation has revealed that codon bias - the uneven use of synonymous codons in the transcriptome - serves as a secondary genetic code: a code that guides the efficiency of protein production, the fidelity of translation and the metabolism of mRNAs. Recent advancements in our understanding of mRNA decay have revealed a tight coupling between ribosome dynamics and the stability of mRNA transcripts; this coupling integrates codon bias into the concept of codon optimality, or the effects that specific codons and tRNA concentrations have on the efficiency and fidelity of the translation machinery. In this Review, we first discuss the evidence for codon-dependent effects on translation, beginning with the basic mechanisms through which translation perturbation can affect translation efficiency, protein folding and transcript stability. We then discuss how codon effects are leveraged by the cell to tailor the proteome to maintain homeostasis, execute specific gene expression programmes of growth or differentiation and optimize the efficiency of protein production.

/pdf/codon-optimality-bias-and-usage-in-translation-and-mrna-59wjdhjlyc.pdf

Codon optimality, bias and usage in translation and mRNA decay.

https://www.cell.com/molecular-cell/pdf/S1097-2765(13)00326-2.pdf

eIF5A promotes translation of polyproline motifs.

High-Precision Analysis of Translational Pausing by Ribosome Profiling in Bacteria Lacking EFP

Clarifying the Translational Pausing Landscape in Bacteria by Ribosome Profiling.

Although the ribosome is a very general catalyst, it cannot synthesize all protein sequences equally well For example, ribosomes stall on the secretion monitor (SecM) leader peptide to regulate expression of a downstream gene Using a genetic selection in Escherichia coli, we identified additional nascent peptide motifs that stall ribosomes Kinetic studies show that some nascent peptides dramatically inhibit rates of peptide release by release factors We find that residues upstream of the minimal stalling motif can either enhance or suppress this effect In other stalling motifs, peptidyl transfer to certain aminoacyl-tRNAs is inhibited In particular, three consecutive Pro codons pose a challenge for elongating ribosomes The translation factor elongation factor P, which alleviates pausing at polyproline sequences, has little or no effect on other stalling peptides The motifs that we identified are underrepresented in bacterial proteomes and show evidence of stalling on endogenous E coli proteins

https://www.pnas.org/content/pnas/110/10/E878.full.pdf

Christopher J. Woolstenhulme

Papers

eIF5A promotes translation of polyproline motifs.

High-Precision Analysis of Translational Pausing by Ribosome Profiling in Bacteria Lacking EFP

Clarifying the Translational Pausing Landscape in Bacteria by Ribosome Profiling.

Nascent peptides that block protein synthesis in bacteria

Genetic Identification of Nascent Peptides That Induce Ribosome Stalling