Biology subjects

Crawford, R. A.

Publications and source records attributed to Crawford, R. A..

2 recordsLinked to original sources

bioRxiv2024

The 3'-untranslated regions of yeast ribosomal protein mRNAs determine paralog incorporation into ribosomes and recruit factors necessary for specialized functions

Ribosome heterogeneity arises via the differential incorporation of ribosomal protein (RP) paralogs, post-transcriptionally modified rRNA, post-translationally modified RPs, and ribosome-associated proteins (RAPs) into ribosomes. This has led to the hypothesis that heterogeneous or "specialized" ribosomes, which translate specific mRNA subsets, confer key roles in cell growth and development. While proven examples of functional ribosome heterogeneity in eukaryotes exist, there is no comprehensive analysis of specialized ribosome formation. We employed yeast RP paralog deletion libraries and high-throughput screening to investigate the functional specificity and redundancy between paralogs under various growth conditions. Composition and translatome analyses verified paralog specificity in the assembly and function of ribosomes specialized for growth on different carbon sources, and identified a novel RAP required for the efficient translation of peroxisomal proteins. Importantly, we also show that the mechanism by which specific RP paralogs incorporate into ribosomes requires their unique 3-untranslated regions to yield ribosomes that differ in composition and function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/585503v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@15ba935org.highwire.dtl.DTLVardef@c210b7org.highwire.dtl.DTLVardef@d77b7dorg.highwire.dtl.DTLVardef@ec72f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Cytosolic aspartate aminotransferase moonlights as a ribosome binding modulator of Gcn2 activity during oxidative stress

Regulation of translation is a fundamental facet of the cellular response to rapidly changing external conditions. Specific RNA-binding proteins (RBPs) co-ordinate the translational regulation of distinct mRNA cohorts during stress. To identify RBPs with previously under-appreciated roles in translational control, we used polysome profiling and mass spectrometry to identify and quantify proteins associated with translating ribosomes in unstressed yeast cells and during oxidative stress and amino acid starvation, which both induce the integrated stress response (ISR). Over 800 proteins were identified across polysome gradient fractions, including ribosomal proteins, translation factors and many others without previously described translation-related roles, including numerous metabolic enzymes. We identified variations in patterns of polysome enrichment in both unstressed and stressed cells and identified proteins enriched in heavy polysomes during stress. Genetic screening of polysome-enriched RBPs identified the cytosolic aspartate aminotransferase, Aat2, as a ribosome-associated protein whose deletion conferred growth sensitivity to oxidative stress. Loss of Aat2 caused aberrantly high activation of the ISR via enhanced eIF2 phosphorylation and GCN4 activation. Importantly, non-catalytic AAT2 mutants retained polysome association and did not show heightened stress sensitivity. Aat2 therefore has a separate ribosome-associated translational regulatory or moonlighting function that modulates the ISR independent of its aspartate aminotransferase activity.

biochemistry↗