Global phosphoproteomics pinpoints uncharted Gcn2-mediated mechanisms of translational control.

The conserved Gcn2 protein kinase mediates cellular adaptations to amino acid limitation through translational control of gene expression that is exclusively executed by phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α). Using quantitative phosphoproteomics, however, we discovered that Gcn2 targets auxiliary effectors to modulate translation. Accordingly, Gcn2 also phosphorylates the ß-subunit of the trimeric eIF2 G protein complex to promote its association with eIF5, which prevents spontaneous nucleotide exchange on eIF2 and thereby restricts the recycling of the initiator methionyl-tRNA-bound eIF2-GDP ternary complex in amino-acid-starved cells. This mechanism contributes to the inhibition of translation initiation in parallel to the sequestration of the nucleotide exchange factor eIF2B by phosphorylated eIF2α. Gcn2 further phosphorylates Gcn20 to antagonize, in an inhibitory feedback loop, the formation of the Gcn2-stimulatory Gcn1-Gcn20 complex. Thus, Gcn2 plays a substantially more intricate role in controlling translation initiation than hitherto appreciated.

Disome and Trisome Profiling Reveal Genome-wide Targets of Ribosome Quality Control.

The ribosome-associated protein quality control (RQC) system that resolves stalled translation events is activated when ribosomes collide and form disome, trisome, or higher-order complexes. However, it is unclear whether this system distinguishes collision complexes formed on defective mRNAs from those with functional roles on endogenous transcripts. Here, we performed disome and trisome footprint profiling in yeast and found collisions were enriched on diverse sequence motifs known to slow translation. When 60S recycling was inhibited, disomes accumulated at stop codons and could move into the 3' UTR to reinitiate translation. The ubiquitin ligase and RQC factor Hel2/ZNF598 generally recognized collisions but did not induce degradation of endogenous transcripts. However, loss of Hel2 triggered the integrated stress response, via phosphorylation of eIF2α, thus linking these pathways. Our results suggest that Hel2 has a role in sensing ribosome collisions on endogenous mRNAs, and such events may be important for cellular homeostasis.

General control non-repressible 20 (GCN20) functions in root growth by modulating DNA damage repair in Arabidopsis.

BACKGROUND: Most ABC transporters are engaged in transport of various compounds, but its subfamily F lacks transmembrane domain essential for chemical transportation. Thus the function of subfamily F remains further elusive. RESULTS: Here, we identified General Control Non-Repressible 20 (GCN20), a member of subfamily F, as new factor for DNA damage repair in root growth. While gcn20-1 mutant had a short primary root with reduced meristem size and cell number, similar primary root lengths were assayed in both wild-type and GCN20::GCN20 gcn20-1 plants, indicating the involvement of GCN20 in root elongation. Further experiments with EdU incorporation and comet assay demonstrated that gcn20-1 displays increased cell cycle arrest at G2/M checkpoint and accumulates more damaged DNA. This is possible due to impaired ability of DNA repair in gcn20-1 since gcn20-1 seedlings are hypersensitive to DNA damage inducers MMC and MMS compared with the wild type plants. This note was further supported by the observation that gcn20-1 is more sensitive than the wild type when subjected to UV treatment in term of changes of both fresh weight and survival rate. CONCLUSIONS: Our study indicates that GCN20 functions in primary root growth by modulating DNA damage repair in Arabidopsis. Our study will be useful to understand the functions of non-transporter ABC proteins in plant growth.

Enhancement and mapping of tolerance to salt stress and 5-fluorocytosine in synthetic yeast strains via SCRaMbLE.

Varied environmental stress can affect cell growth and activity of the cellular catalyst. Traditional path of adaptive evolution generally takes a long time to achieve a tolerance phenotype, meanwhile, it is a challenge to dissect the underlying genetic mechanism. Here, using SCRaMbLE, a genome scale tool to generate random structural variations, a total of 222 evolved yeast strains with enhanced environmental tolerances were obtained in haploid or diploid yeasts containing six synthetic chromosomes. Whole genome sequencing of the evolved strains revealed that these strains generated different structural variants. Notably, by phenotypic-genotypic analysis of the SCRaMbLEd strains, we find that a deletion of gene YFR009W (GCN20) can improve salt tolerance of Saccharomyces cerevisiae, and a deletion of gene YER056C can improve 5-flucytosine tolerance of Saccharomyces cerevisiae. This study shows applications of SCRaMbLE to accelerate phenotypic evolution for varied environmental stress and to explore relationships between structural variations and evolved phenotypes.

Ribo-Seq and RNA-Seq of TMA46 ( DFRP1) and GIR2 ( DFRP2) knockout yeast strains.

In eukaryotes, stalled and collided ribosomes are recognized by several conserved multicomponent systems, which either block protein synthesis in situ and resolve the collision locally, or trigger a general stress response. Yeast ribosome-binding GTPases RBG1 (DRG1 in mammals) and RBG2 (DRG2) form two distinct heterodimers with TMA46 (DFRP1) and GIR2 (DFRP2), respectively, both involved in mRNA translation. Accumulated evidence suggests that the dimers play partially redundant roles in elongation processivity and resolution of ribosome stalling and collision events, as well as in the regulation of GCN1-mediated signaling involved in ribosome-associated quality control (RQC). They also genetically interact with SLH1 (ASCC3) helicase, a key component of RQC trigger (RQT) complex disassembling collided ribosomes. Here, we present RNA-Seq and ribosome profiling (Ribo-Seq) data from S. cerevisiae strains with individual deletions of the TMA46 and GIR2 genes. Raw RNA-Seq and Ribo-Seq data as well as gene-level read counts are available in NCBI Gene Expression Omnibus (GEO) repository under GEO accession GSE185458 and GSE185286.