Regulation of protein Translation

Regulation of protein translation


Principle investigators: Dr. Johannes Hanson and Dr. Leonie Bentsink (Wageningen UR)

The ribosome. Proteins (blue) and RNA (red and light blue).

Translation, the synthesis of proteins using mRNA as template, is a fundamental biochemical process. It is by far the most energy consuming process of the cell. Interestingly, it is highly regulated in response to changes in energy availability. This regulatory circuit is conserved in all eukaryotes from man to yeast and tightly linked to the regulation of growth. In plants, we are studying this process since it bears promise for future crop yield improvement.

We specifically study three aspects of translational regulation in Arabidopsis.

I. The heterogeneous ribosome as a regulatory hub.
Proteomic characterization of ribosomal protein changes.

The protein translation machine, the ribosome, is the most complex protein complex of the cell, even visible by the electron microscope. It consists of 79 proteins and four RNA molecules. We have show that different iso-forms of the ribosomal proteins are present in the ribosome in cells with high energy availability compared to starving cells. Global translational patterns change according to the energy level of the cell. Thousands of peptides derived from purified Arabidopsis ribosomes were analyzed by MSE MS-MS analysis and on a global level the levels from sugar treated plants (see figure; red) are clearly different than levels from control samples (green) (The two components shown represent 48% of total variation). Panel B in the Figure represents the levels of several identified ribosomal proteins show significant changes.

We are currently approaching the dynamic ribosome using genetic tools combined with proteomic and microarray analysis to characterize the signaling pathway in molecular detail.

II Translation of stored RNA in the germinating seed.
Transcription is not needed during the first phases of germination (Figure from Rajjou et al, 2004, Plant Physiology).

Seeds do not photosynthesize and depend on the stored energy reserves during germination and seedling establishment. The proteins needed for the early phases of germination are present in the dry seed together with many mRNAs, which are presumably the first to be translated following germination. Recent proteome and transcriptome data have revealed the importance of translational regulation both at the global level and at the level of individual transcripts.

This project addresses the importance of seed stored mRNAs for regulation of dormancy and germination, by i.e. analyzing the translatome of dormant and after-ripened seeds. Part of the project will be based on translation related genes of which the expression is affected in highly dormant seeds.

III Sucrose induced inhibition of translation
Our current model. The present datasets support a model in which the peptide encoded by the uORF cause stalling of ribosomes on the bZIP mRNA and thereby inhibiting translation of the downstream transcription factor encoding ORF.

Five bZIP transcription factors (the S1 group) shown to be capable of reprogramming metabolism are regulated on the translational level in response to sucrose concentrations. We have shown this to be dependent on upstream open reading frames (uORFs) present in the 5’leaders of the S1 bZIP mRNAs. We are now identifying the factors that are involved in the regulation and thereby gain information on the signaling pathway regulating this process. We use biochemistry, genetics and molecular genetics. We are currently using yeast two hybrid analysis, immuno-precipitations combined with label free LC-MS (proteomics), expression analysis in protoplasts, combined with transgenic and mutant plants as tools in our investigations.