This project investigated mechanisms that regulate the translation of genetic information from mRNA into proteins using experimental and computational approaches. Experimentally, the work focused on translational control in the plant reference species Arabidopsis thaliana. One arm of the project focused on the coordination between protein synthesis and the cell cycle in the root stem cell niche. A second arm investigated the role of a pan-eukaryotic signaling pathway in adapting plant protein synthesis to environmental stress conditions. The project also advanced several computational models of the translation process that opened new insights into the evolution of codon usage and the robustness of translation against ribosome decoding errors.

Outcomes, intellectual merit:

1. In the aerial part of the plant, the GCN2 protein kinase was identified as linking protein synthesis in the cytosol to the photosynthetic status of the chloroplast. Specifically, reactive oxygen species from the chloroplast can activate GCN2 in the cytosol, a potential retrograde signaling event whereby the chloroplast feeds back on cytosolic protein synthesis.

2. The role of GCN2 kinase in modulating the translatome as well as the transcriptome of Arabidopsis seedlings was described under herbicide stress.

3. The GCN2 kinase contributes to plant growth and development under high light, salt and cold stress, in keeping with the hypothesis that GCN2 functions as a sensor for reactive oxygen.

4. In the root, the EBP1 protein (ErbB3 binding protein) has the remarkable ability to curtail the repression of the cell cycle, a function of the Retinoblastoma-related (RBR) protein of Arabidopsis. This finding by partners on this project at the University of London was funded by a linked award from the British government.

5. EBP1 has been implicated in ribosome biogenesis, a finding that was confirmed here for Arabidopsis by studying the subcellular localization of EBP1 as well as its role in ribosomal RNA processing.

6. Technology for measuring the efficiency of translation was advanced by adapting the PuNCH-P technique (Puromycin associated nascent chain proteomics) for use in plants.

7. We created mathematical models to infer the rate with which ribosomes generate processivity errors during translation elongation. These errors can now be estimated in a codon-specific manner from ribosome footprinting data and, independently, from position- and gene-specific patterns of synonymous codon usage. We are also close to completing the implementation of both models within a previously developed R-based software package, AnaCoDa.  Models are Bayesian in nature and, as a result, the models can incorporate additional information on gene expression and mutation bias.

Outcomes, broader impacts:

The project supported the professional development of one postdoctoral associate and five graduate students two of whom have since graduated with a PhD and one with an MS. Three additional first year graduate students took part in lab rotations. Ten undergraduate students received training by participating in the research; of these, four individuals coauthored publications and three helped to develop code. One personnel with Hispanic and two with Middle Eastern heritage, ethnicities broadly underrepresented in US science, participated in the project. 

Overall, these findings advance the mechanistic understanding of how the central process of protein synthesis is coordinated with both environmental conditions and developmental processes controlling cell division. The quantitative and computational representations of these complex events raise the likelihood that protein synthesis will lend itself to rational and predictable redesign in the incipient age of synthetic biology.

Last Modified: 10/29/2020
Modified by: Albrecht G Von Arnim