Sucrose-mediated translational stalling involves a conserved ribosomal pocket

Within eukaryotes, 20-50% of the mRNAs contain short open reading frames (uORFs) located upstream of the main ORF. A significant fraction of these uORFs encode conserved peptides (CPuORFs) that regulate translation in response to specific metabolites. A well-studied example includes uORF2 of the plant growth inhibiting transcription factor bZIP11. Elevated intracellular sucrose levels lead to ribosome stalling at the stop codon of uORF2, thus reducing bZIP11 protein synthesis. Similar examples can be found in bacteria and animals, e.g. on the bacterial TnaC and human CDH1-NPN* ORFs that both induce stalling at the stop codon when in the presence of tryptophan and the drug-like molecule PF846, respectively. In this study, we affinity-purified in vitro translated sucrose-stalled wheat ribosomes translating bZIP11-uORF2 and determined the ribosomes’ structures using cryo-electron microscopy. This revealed density inside a pocket in the ribosomal exit tunnel of the plant Triticum aestivum, that colocalizes with the binding locations of tryptophan and PF846 in E. coli and humans, respectively. We suggest this density corresponds to sucrose. Tryptophan and PF846 mode-of-action was previously proposed to inhibit release factor binding or function. Mutation of the uORF2 stop codon shows that its presence is crucial for sucrose-induced stalling, suggesting that the stalling only manifests during termination and not elongation. Moreover, the structural similarities with tryptophan-induced stalled ribosomes near the peptidyl transferase center indicates that an analogous mechanism of inhibition of release factor function is likely. Our findings suggest a conserved mechanistic framework across different organisms, wherein specific molecules interact with the nascent peptide and ribosome to modulate protein synthesis

Metabolite control of protein translation mediated by Conserved Peptide uORFs

Maintaining homeostasis during fluctuating conditions is crucial for plant development, survival and reproductive success. Evolutionary forces provided plants with a complex network of regulatory pathways to maintain homeostasis and prevent starvation. Part of this network is regulated by controlling the mRNA translation of key genes in (energy) metabolism via upstream open reading frames (uORFs). In chapter one energy metabolisms and general principles of protein translation and its regulation are explained. In chapter two, the role of uORFs with conserved peptide sequences (CPuORFs) on translation regulation is described in detail. Several CPuORFs can control stalling of ribosomes upon the presence of a metabolite, which is specific to the amino acid sequence of the CPuORF. For example, sucrose induces ribosomal stalling on a CPuORF present on the mRNA of bZIP11 and other S1 group bZIPs. A handful of CPuORFs were experimentally confirmed. We were curious as to whether more of such CPuORFs are present in the Arabidopsis thaliana genome. Therefore, in chapter three a bioinformatical pipeline was developed to search for CPuORFs, unbiased for start codon. This pipeline utilized well-annotated sequence data from 31 eudicot plant species to search for amino acid conservation in the 5’leaders. 29 novel CPuORFs were identified, of which fifteen did not possess an AUG start codon. In chapter four, three of the bioinformatically predicted CPuORFs, present in the mRNAs of the energy signaling genes SNAK2, TPPG and Raptor1, were tested on functionality. First we showed that the CPuORFs of SNAK2 and Raptor1 induce ribosome stalling when translated in vitro. Next, 25 metabolites were tested for influence on CPuORF regulated translation, by developing a luciferase-based in vitro translation assay. The mechanism of metabolite-induced ribosome stalling on CPuORFs remains unclear. Therefore, in chapter five we explored the function of the bZIP11 CPuORF and discovered that the position of the stop codon is crucial in the angiosperm CPuORFs, but not in gymnosperm CPuORFs. To further unravel the mechanisms of sucrose-induced ribosome stalling on the bZIP11 CPuORF, stalled ribosomes were purified in chapter six. Next, single particle cryo-EM was performed on the purified ribosomes to determine its structure. This resulted in a high-resolution structure of the wheat germ ribosome. Interestingly, the release factor was absent in the structure indicating that the ribosome does not allow for release factor binding. In chapter seven, we wanted to investigate sucrose-induced ribosome stalling on the bZIP11 CPuORF in vivo. Ribosome profiling on Arabidopsis seedlings overexpressing mRNA with wild type or mutated bZIP11 CPuORF confirmed that stalling occurs at the stop codon and that a single amino acid mutation can abolish ribosome stalling. Next, we aimed to purify these stalling ribosomes by purifying the mRNA using a catalytically inactive Cas9 (dCas9). Finally, a model for metabolite sensing by the ribosome and CPuORF is discussed in chapter eight. Moreover, this chapter covers the role of CPuORFs across different domains of life, potential applications of CPuORFs and discusses the future direction of protein translation research

Metabolite control of protein translation mediated by Conserved Peptide uORFs

Maintaining homeostasis during fluctuating conditions is crucial for plant development, survival and reproductive success. Evolutionary forces provided plants with a complex network of regulatory pathways to maintain homeostasis and prevent starvation. Part of this network is regulated by controlling the mRNA translation of key genes in (energy) metabolism via upstream open reading frames (uORFs). In chapter one energy metabolisms and general principles of protein translation and its regulation are explained. In chapter two, the role of uORFs with conserved peptide sequences (CPuORFs) on translation regulation is described in detail. Several CPuORFs can control stalling of ribosomes upon the presence of a metabolite, which is specific to the amino acid sequence of the CPuORF. For example, sucrose induces ribosomal stalling on a CPuORF present on the mRNA of bZIP11 and other S1 group bZIPs. A handful of CPuORFs were experimentally confirmed. We were curious as to whether more of such CPuORFs are present in the Arabidopsis thaliana genome. Therefore, in chapter three a bioinformatical pipeline was developed to search for CPuORFs, unbiased for start codon. This pipeline utilized well-annotated sequence data from 31 eudicot plant species to search for amino acid conservation in the 5’leaders. 29 novel CPuORFs were identified, of which fifteen did not possess an AUG start codon. In chapter four, three of the bioinformatically predicted CPuORFs, present in the mRNAs of the energy signaling genes SNAK2, TPPG and Raptor1, were tested on functionality. First we showed that the CPuORFs of SNAK2 and Raptor1 induce ribosome stalling when translated in vitro. Next, 25 metabolites were tested for influence on CPuORF regulated translation, by developing a luciferase-based in vitro translation assay. The mechanism of metabolite-induced ribosome stalling on CPuORFs remains unclear. Therefore, in chapter five we explored the function of the bZIP11 CPuORF and discovered that the position of the stop codon is crucial in the angiosperm CPuORFs, but not in gymnosperm CPuORFs. To further unravel the mechanisms of sucrose-induced ribosome stalling on the bZIP11 CPuORF, stalled ribosomes were purified in chapter six. Next, single particle cryo-EM was performed on the purified ribosomes to determine its structure. This resulted in a high-resolution structure of the wheat germ ribosome. Interestingly, the release factor was absent in the structure indicating that the ribosome does not allow for release factor binding. In chapter seven, we wanted to investigate sucrose-induced ribosome stalling on the bZIP11 CPuORF in vivo. Ribosome profiling on Arabidopsis seedlings overexpressing mRNA with wild type or mutated bZIP11 CPuORF confirmed that stalling occurs at the stop codon and that a single amino acid mutation can abolish ribosome stalling. Next, we aimed to purify these stalling ribosomes by purifying the mRNA using a catalytically inactive Cas9 (dCas9). Finally, a model for metabolite sensing by the ribosome and CPuORF is discussed in chapter eight. Moreover, this chapter covers the role of CPuORFs across different domains of life, potential applications of CPuORFs and discusses the future direction of protein translation research

Translational dynamics during seed maturation

We analysed the polysomal mRNA and total mRNA during Col-0 seed maturation The aim was to investigate the translational dynamics during seed maturatio

Novel pipeline identifies new upstream ORFs and non-AUG initiating main ORFs with conserved amino acid sequences in the 5' leader of mRNAs in Arabidopsis thaliana

Eukaryotic mRNAs contain a 5' leader preceding the main open reading frame (mORF) and, depending on the species, 20-50% of eukaryotic mRNAs harbor an upstream ORF (uORF) in the 5' leader. An unknown fraction of these uORFs encode sequence conserved peptides (conserved peptide uORFs, CPuORFs). Most experimentally validated CPuORFs demonstrated to regulate the translation of the downstream main ORF, usually in a metabolite concentration dependent manner. To this end, comparative genomic approaches have been used to identify novel CPuORFs, by comparing AUG initiating uORF sequences of the Arabidopsis genome or Arabidopsis ESTs. Previous research has shown that most CPuORFs possess a start codon context suboptimal for translation initiation, which turns out to be favorable for translational regulation. The suboptimal initiation context may even include non-AUG start codons, which makes CPuORFs hard to predict. For this reason, we developed a novel pipeline to identify CPuORFs unbiased of start codon using well annotated sequence data from 32 eudicot plant species and rice. Our new pipeline was able to identify 30 novel Arabidopsis CPuORFs, conserved across a wide variety of eudicot species of which 16 do not initiate with an AUG start codon. In addition to CPuORFs, the pipeline was able to find 14 conserved coding regions directly upstream and in frame with the main ORF, which likely initiate translation on a non-AUG start codon. Altogether, our pipeline identified highly conserved coding regions in the 5' leaders of Arabidopsis transcripts, including in genes with proven functional importance such as LHY, a key regulator of the circadian clock, and the RAPTOR1 subunit of the Target Of Rapamycin (TOR) kinase

Translational dynamics during seed maturation

We analysed the polysomal mRNA and total mRNA during Col-0 seed maturation The aim was to investigate the translational dynamics during seed maturatio

Translational dynamics during seed maturation

We analysed the polysomal mRNA and total mRNA during Col-0 seed maturation The aim was to investigate the translational dynamics during seed maturatio

Metabolite Control of Translation by Conserved Peptide uORFs: The Ribosome as a Metabolite Multi-sensor

The regulation of gene expression is intensely investigated in diverse biological systems. Gene expression involves RNA transcription, RNA splicing, RNA stability, translation, posttranslational modification and protein stability. Particular attention has been given to mRNA levels due to advances in microarray analysis and RNA-sequencing techniques. However, transcript levels do not necessarily correlate with protein levels or functionality (Conrads et al., 2005; Gibon et al., 2006; Bianchini et al., 2008) and complex layers of posttranscriptional regulation have been uncovered, foremost mRNA translation. Translation can be regulated both globally and in a transcript-specific manner. Examples of global mRNA translational regulation include availability of ribosomes, and translation initiation, elongation and termination factors. In transcript-specific translational regulation individual mRNA species or mRNA groups are selectively translated. For example, mRNAs can be sequestered in stress granules, removing them from the translatable mRNA pool (Chantarachot and Bailey-Serres, 2017). mRNA sequence or structural features can affect translatability directly or indirectly, the latter via small RNAs or mRNA binding proteins (reviewed in Merchante et al., 2017). Upstream open reading frames (uORFs) have been shown to participate in both global and transcript-specific regulation (Von Arnim et al., 2014). Here, recent advances in translation regulation by uORFs are discussed, focusing on uORFs encoding sequence conserved peptides (CPuORFs)