Abstract
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
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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.
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