Refining the composition of the Arabidopsis thaliana 80S cytosolic ribosome

Abstract

The cytosolic 80S ribosome is composed of protein and RNA molecules and its function in protein synthesis is modulated through interaction with other cytosolic components. Defining the role of each of the proteins associated with ribosomes in plants is a major challenge which is hampered by difficulties in attribution of different proteins to roles in ribosome biogenesis, the mature cytosolic ribosome (r-proteins) or to the broader translatome associated with functioning ribosomes. Here we refined the core r-protein composition in plants by determining the abundance of proteins in low, partially and highly purified ribosomal samples from Arabidopsis thaliana cell cultures. To characterise this list of proteins further we determined their degradation (KD) and synthesis (KS) rate by progressive labelling with 15 N combined with peptide mass spectrometry analysis. The turnover rates of 55 r-proteins, including 26 r-proteins from the 40S subunit and 29 r-proteins from the 60S subunit could be determined. Overall, ribosome proteins showed very similar KD and KS rates suggesting that half of the ribosome population is replaced every 3-4 days. Three proteins showed significantly shorter half-lives; ribosomal protein P0D (RPP0D) with a half-life of 0.5 days and RACK1b and c with half-lives of 1-1.4 days. The ribosomal RPP0D protein is a homolog of the human Mrt4 protein, a trans-acting factor in the assembly of the pre-60S particle, while RACK1 has known regulatory roles in cell function beyond its role as a 40S subunit. Our experiments also identified 58 proteins that are not from r-protein families but co-purify with ribosomes and co-express with rproteins in Arabidopsis. Of this set, 26 were enriched more than 10-fold during ribosome purification. A number have known roles in translation or ribosome-association while others are newly proposed ribosome-associated factors in plants. This analysis provides a more robust understanding of Arabidopsis ribosome content, shows that most r-proteins turnover in unison in vivo, identifies a novel set of potential plant translatome components, and reveals how protein turnover can identify r-proteins involved in ribosome biogenesis or regulation in plants. Data are available via ProteomeXchange with identifier PXD012839.

Arabidopsis REI-LIKE proteins activate ribosome biogenesis during cold acclimation

“…The requirement of de novo synthesis in the cold is evident by slow recovery of ribosome complex abundances in the reil mutants. The time scale of 3-7 days after the shift is in agreement with previous reports of ribosome protein turnover in Arabidopsis (Li et al 2017; Salih et al, 2019), where half-life of the ribosome population was approximately 3-4 days. Only RPP0D and the RACK1B and 1C paralogs were shorter lived with half-lives between 0.5 and 1.5 days (Salih et al, 2019).…”

“…The time scale of 3-7 days after the shift is in agreement with previous reports of ribosome protein turnover in Arabidopsis (Li et al 2017; Salih et al, 2019), where half-life of the ribosome population was approximately 3-4 days. Only RPP0D and the RACK1B and 1C paralogs were shorter lived with half-lives between 0.5 and 1.5 days (Salih et al, 2019). Induction of 40S and 60S RP transcripts after cold shift in the Col-0 wild type and compensatory enhanced and prolonged expression of these genes in reil1 reil2 double mutants indicate that Arabidopsis roots ( Fig.…”

Systematic Review of Plant Ribosome Heterogeneity and Specialization

“…The striking difference in the detected paralogs per RP family in both studies may be explained by technical variation of the complex CRP preparation and proteomic analysis but may equally likely originate from changes of ribosome heterogeneity between the two investigated ribosome populations. The most recent study that refined the compositions of the Arabidopsis cytosolic ribosome mapped 76 RP families and 184 members using a label-assisted proteomics approach ( Salih et al., 2019 ).…”

“…These studies suggest that a potential mechanism of ribosome remodeling exists even though RPs are in general stable and long-lived ( Li et al., 2017a ). Ribosomal complexes of Arabidopsis have a mean-RP half-life of 3-4 days ( Salih et al., 2019 ). Considering the general stability of ribosomes, it seems likely that the high variation among RPs results from induced translation targeted to specific RPs or RP paralogs and remodeling of pre-existing ribosome complexes by RP exchange.…”

The Consequences of a Disruption in Cyto-Nuclear Coadaptation on the Molecular Response to a Nitrate Starvation in Arabidopsis

“…Plant cells are known to coordinate eukaryotic ribosomal protein (cyto-ribosome) and prokaryotic ribosomal protein (chloro-ribosome and mito-ribosome) productions [96]. The composition, structure and organization of the three cellular ribosomes have been studied in detail in Arabidopsis [97][98][99]. We listed 245 genes encoding cyto-ribosome RPs, 66 genes for chloro-ribosome and 93 genes for the mito-ribosome proteins (Table S10).…”