Biology subjects

Barros, G. C.

Publications and source records attributed to Barros, G. C..

3 recordsLinked to original sources

bioRxiv2024

Context specific ubiquitin modification of ribosomes regulate translation under oxidative stress

Cellular exposure to stress is known to activate several translational control pathways through ribosome ubiquitination. However, how unique patterns of ribosome ubiquitination act at the site-specific level to drive distinct modes of translation regulation remains unclear. To further understand the complexity of these ubiquitin signals, we developed a new targeted proteomics approach to quantify site-specific ubiquitin modification across the ribosome. This method increased the sensitivity and throughput of current approaches and allowed us to systematically measure the ubiquitin status of 78 ribosome peptides and ubiquitin linkages in response to stress. Using this method, we were able to detect the ubiquitination of several ribosome sites even in steady-state conditions, and to show that their modification increases non-stoichiometrically in a dynamic range of >4 orders of magnitude in response to hydrogen peroxide. Besides demonstrating new patterns of global ribosome ubiquitination, our study also revealed an unexpected increase of ubiquitination of ribosomal protein uS10/Rps20 and uS3/Rps3 independent of the canonical E3 ubiquitin ligase Hel2. Furthermore, we show that unique and mixed patterns of ribosome ubiquitination occur in a stress specific manner, depending on the nature of stressor and the enzymes involved. Finally, we showed that while deletion of HEL2 further induces the integrated stress response in response to the nucleotide alkylating agent 4-NQO, deletion of the E2 conjugase RAD6 leads to sustained translation only in response to H2O2. Our findings contribute to deciphering the complexity of the stress response at the translational level, revealing the induction of dynamic and selective ubiquitin codes, which shed light on the integration of important quality control pathways during cellular response to stress.

molecular biology↗

bioRxiv2019

Endogenous polybasic peptides cause slower movement of ribosomes but are not targets of the ribosome quality control complex (RQC)

Highly positively charged protein segments are known to result in poor translation efficiency. This effect is explained by ribosome stalling caused by electrostatic interactions between the nascent peptide and the negatively charged ribosome exit tunnel, leading to translation termination followed by protein degradation mediated by the RQC complex. These polybasic sequences are mainly studied in the context of artificial reporter systems. Examples of endogenous yeast proteins targeted by the RQC complex are Rqc1, a protein essential for RQC function, and Sdd1. Both contain polybasic sequences that are thought to activate the RQC, leading to protein down-regulation. Here, we investigated whether the RQC complex regulates other endogenous proteins with polybasic sequences. We show by bioinformatics, ribosome profiling data analysis, and western blot that endogenous proteins containing polybasic sequences similar to, or even more positively charged than those of Rqc1 and Sdd1, are not targeted by the RQC complex suggesting that endogenous polybasic sequences are not sufficient to induce this type of regulation. Finally, our results also suggest that Rqc1 is regulated post-translationally by the E3 component of the RQC complex Ltn1, in a manner independent of the RQC complex.

biochemistry↗

bioRxiv2019

The distribution, conservation and correlation with translational efficiency of the first five codons in Escherichia coli genes

Translation initiation is a critical step in the regulation of protein synthesis, and it is subjected to different control mechanisms, such as 5 UTR secondary structure and initiation codon context, that can influence the rates at which initiation and consequentially translation occur. For some genes, translation elongation also affects the rate of protein synthesis. With a GFP library containing nearly all possible combinations of nucleotides from the 3rd to the 5th codon positions in the protein coding region of the mRNA, it was previously demonstrated that some nucleotide combinations increased GFP expression up to four orders of magnitude. While it is clear that the codon region from positions 3 to 5 can influence protein expression levels of artificial constructs, its impact on endogenous proteins is still unknown. Through bioinformatics analysis, we identified the nucleotide combinations of the GFP library in Escherichia coli genes and examined the correlation between the expected levels of translation according to the GFP data with the experimental measures of protein expression. We observed that E. coli genes were enriched with the nucleotide compositions that enhanced protein expression in the GFP library, but surprisingly, it seemed to affect the translation efficiency only marginally. Nevertheless, our data indicate that different enterobacteria present similar nucleotide composition enrichment as E. coli, suggesting an evolutionary pressure towards the conservation of short translational enhancer sequences.

bioinformatics↗