Project Outline

How do plant cells make sure that the proteins they produce are error free and fully functional?

Proteins are essential components of cells, and the proteome - i.e., the complete cohort of proteins and their modification status - must be effectively regulated to maintain cellular integrity. A critical contributory step to proteome function is protein synthesis, where mRNAs are decoded and translated into polypeptides by the ribosome. However, problems can arise during this process, leading to the production of aberrant proteins that negatively impact cellular function. Consequently, defective mRNAs and proteins must be recognised and destroyed, and dedicated machineries exist to facilitate this. Despite their central importance for ensuring proteome homeostasis (proteostasis), mechanisms controlling co-translational mRNA and protein quality in the plant kingdom are still largely unknown. This represents a major ‘black-box’ in the field of plant cell biology, since precise protein translation is essential for coordinating growth, development and resistance to environmental stresses that negatively impact plant productivity and survival. Increasing our understanding of the breadth of mechanisms employed by plants to ensure effective mRNA translation under stress has strong potential to identify novel targets for manipulating traits of agronomic importance and could facilitate the development of improved crop varieties with enhanced performance. As such, this project aligns with the BBSRC priority area of sustainable agriculture and food.

We recently identified a family of E3 ubiquitin ligases in Arabidopsis that function at the interface of mRNA translation and protein destruction. Here, we will investigate the hypothesis that these ligases are components of an expanded and plant-specific “toolkit” that provides dynamic and stress-responsive functions in translational quality control.

Using a range of cutting edge molecular and biochemical techniques, we will decipher roles for these regulators in controlling global translation. There are several key areas where a PhD candidate would focus their investigations:

• Defining how these E3 ligases dynamically associate with ribosomes, proteasomes and other cellular machineries to form ‘translasomes’ that act as quality surveillance hubs.
• Characterising their direct proteolytic and mRNA targets.
• Determining how they collectively contribute to the control of global and stress-responsive mRNA translation and degradation.
• Defining their roles in the proteolytic turnover of stalled or misfolded proteins that form when translational errors arise.

The majority of our work is carried out in the genetic model plant Arabidopsis, with the eventual aim of this research being to translate key findings into crop species, such as barley or rice. The research will be largely molecular based and will also include ‘omics’ approaches. The PhD candidate will therefore gain expertise in a wide range of cutting edge and transferable techniques.

Techniques

• Standard molecular genetics (PCR, qPCR, transgenic generation etc)
• Western Blotting
• Immunoprecipitation
• Yeast two hybrid
• RNAseq and RIP-seq
• Polysome profiling
• mRNA degradome analyses