A proteomics tool capable of identifying single proteins would be important for cell biology research and applications. Here, we demonstrate a nanopore-based single-molecule peptide reader sensitiv...

Multiple rereads of single proteins at single–amino acid resolution using nanopores

Cells of all organisms survey problems during translation elongation, which may happen as a consequence of mRNA aberrations, inefficient decoding, or other sources In eukaryotes, ribosome-associated quality control (RQC) senses elongation-stalled ribosomes and promotes dissociation of ribosomal subunits This so-called ribosomal rescue releases the mRNA for degradation and allows 40S subunits to be recycled for new rounds of translation However, the nascent polypeptide chains remain linked to tRNA and associated with the rescued 60S subunits As a final critical step in this pathway, the Ltn1/Listerin E3 ligase subunit of the RQC complex (RQCc) ubiquitylates the nascent chain, which promotes clearance of the 60S subunit while simultaneously marking the nascent chain for elimination Here we review the ribosomal stalling and rescue steps upstream of the RQCc, where one witnesses intersection with cellular machineries implicated in translation elongation, translation termination, ribosomal subunit recycling, and mRNA quality control We emphasize both recent progress and future directions in this area, as well as examples linking ribosomal rescue with the production of Ltn1-RQCc substrates

Ribosomal Stalling During Translation: Providing Substrates for Ribosome-Associated Protein Quality Control.

A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post-translational modifications. In top-down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top-down proteomic workflows. In this review, some recent advances are outlined and current challenges and future directions for the field are discussed.

Identification and Quantification of Proteoforms by Mass Spectrometry.

Russian Science Foundation (Grant Numbers 14-14-00127 and RBFR 14-04-00412); Science Foundation Ireland (Grant Number 12/IA/1335)

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Insights into the mechanisms of eukaryotic translation gained with ribosome profiling.

Although protein synthesis dynamics has been studied both with theoretical models and by profiling ribosome footprints, the determinants of ribosome flux along open reading frames (ORFs) are not fully understood Combining measurements of protein synthesis rate with ribosome footprinting data, we here inferred translation initiation and elongation rates for over a 1,000 ORFs in exponentially growing wild-type yeast cells We found that the amino acid composition of synthesized proteins is as important a determinant of translation elongation rate as parameters related to codon and transfer RNA (tRNA) adaptation We did not find evidence of ribosome collisions curbing the protein output of yeast transcripts, either in high translation conditions associated with exponential growth, or in strains in which deletion of individual ribosomal protein (RP) genes leads to globally increased or decreased translation Slow translation elongation is characteristic of RP-encoding transcripts, which have markedly lower protein output compared with other transcripts with equally high ribosome densities

https://www.pnas.org/content/pnas/116/30/15023.full.pdf

Protein synthesis rates and ribosome occupancies reveal determinants of translation elongation rates.

As proteins are synthesized, the nascent polypeptide must pass through a negatively charged exit tunnel During this stage, positively charged stretches can interact with the ribosome walls and slow the translation Therefore, charged polypeptides may be important factors that affect protein expression To determine the frequency and distribution of positively and negatively charged stretches in different proteomes, the net charge was calculated for every 30 consecutive amino acid residues, which corresponds to the length of the ribosome exit tunnel The following annotated and reviewed proteins in the UniProt database (Swiss-Prot) were analyzed: 551,705 proteins from different organisms and a total of 180 million protein segments We observed that there were more negative than positive stretches and that super-charged positive sequences (ie, net charges ≥ 14) were underrepresented in the proteomes Overall, the proteins were more positively charged at their N-termini and C-termini, and this feature was present in most organisms and subcellular localizations To investigate whether the N-terminal charges affect the elongation rates, previously published ribosomal profiling data obtained from S cerevisiae, without translation-interfering drugs, were analyzed We observed a nonlinear effect of the charge on the ribosome occupancy in which values ≥ +5 and ≤ -6 showed increased and reduced ribosome densities, respectively These groups also showed different distributions across 80S monosomes and polysomes Basic polypeptides are more common within short proteins that are translated by monosomes, whereas negative stretches are more abundant in polysome-translated proteins These findings suggest that the nascent peptide charge impacts translation and can be one of the factors that regulate translation efficiency and protein expression

/pdf/protein-charge-distribution-in-proteomes-and-its-impact-on-48bpqb87p0.pdf

Protein charge distribution in proteomes and its impact on translation

It has been proposed that polybasic peptides cause slower movement of ribosomes through an electrostatic interaction with the highly negative ribosome exit tunnel. Ribosome profiling data—the sequencing of short ribosome-bound fragments of mRNA—is a powerful tool for the analysis of mRNA translation. Using the yeast Saccharomyces cerevisiae as a model, we showed that reduced translation efficiency associated with polybasic protein sequences could be inferred from ribosome profiling. However, an increase in ribosome density at polybasic sequences was evident only when the commonly used translational inhibitors cycloheximide and anisomycin were omitted during mRNA isolation. Since ribosome profiling performed without inhibitors agrees with experimental evidence obtained by other methods, we conclude that cycloheximide and anisomycin must be avoided in ribosome profiling experiments.

https://www.tandfonline.com/doi/pdf/10.1080/15476286.2016.1172755

Increased ribosome density associated to positively charged residues is evident in ribosome profiling experiments performed in the absence of translation inhibitors

Henrique José Araujo de Souza

Papers

Protein charge distribution in proteomes and its impact on translation

Increased ribosome density associated to positively charged residues is evident in ribosome profiling experiments performed in the absence of translation inhibitors