Discovery of proteins involved in the expression and assembly of specific chloroplast gene products through a novel polysome immunoprecipitation approach
Final Report Abstract
During the three year granting period of the Research Fellowship (including reintegration grant) I achieved several outcomes which led to a better understanding of chloroplast translation and the processes contributing to its regulation. Most strikingly, by modification of the ribosome profiling method to an array-based approach we developed and established an efficient method to analyze chloroplast translation at high resolution and in a time and cost effective manner. Using this method I started the high-throughput screen of the maize photosynthetic mutant library in the Barkan lab for specific defects in chloroplast translation. By doing so we discovered the function of PPR53, a chloroplast PPR-SMR protein, as a translational activator of the ndhA-mRNA and factor involved in processing of a 23S-rRNA precursor. Through the analysis of numerous other mutants by array-based ribosome profiling we revealed some basic principles in the translational behavior of chloroplast ribosomes: (i) We found that in intron containing genes translation initiates and elongates independent from the splicing status of the introns. This indicates that chloroplasts did not evolve mechanisms to temporarily or spatially separate translation from splicing as exemplified by nucleus-cytoplasm organization of splicing and translation in eukaryotes. (ii) We found specific pausing sites for elongating chloroplast ribosomes which are probably caused by base pairing of the 16S-rRNA anti-Shine-Dalgarno sequence with Shine-Dalgarno like sequences within open reading frames. This mechanism is similar to ribosome pausing detected in bacteria and might have some implications for co-translational processes as protein folding, assembly, modification, and targeting. (iii) We found no translational coupling for the pair of overlapping atpB/E reading frames as it is typical for most overlapping reading frames in bacteria and was also proposed for chloroplasts. Furthermore the analysis of chloroplast translation in two independent atpB-translation knockout lines clearly demonstrated that, in contrast to Chlamydomonas, in land plant chloroplasts no translational cross-regulation (i.e. control by epistasis of synthesis – CES) initiates from sensing of the AtpB expression status. In addition, a modification of the method enabled us to study the suborganellar localization of translation at high resolution revealing that thylakoid integral proteins are only co-translationally inserted into the thylakoids if the interspace between the first transmembrane domain and the stop codons exceeds a certain distance. Altogether, array-based ribosome profiling has the potential to replace some classic methods in mutant screens for chloroplast translation defects (e.g. polysome-studies, pulse labeling experiments, Northerns) and the approach is likely to be useful for studies of mitochondrial gene expression as well.
Publications
- Mutation of the pentatricopeptide repeat-SMR protein SVR7 impairs accumulation and translation of chloroplast ATP synthase subunits in Arabidopsis thaliana. J Plant Res, 2012, 126 [3], 403-414
Zoschke, R.; Qu, Y.; Zubo, Y.O.; Börner, T.; Schmitz-Linneweber, C.
(See online at https://doi.org/10.1007/s10265-012-0527-1) - The pentatricopeptide repeat-SMR protein ATP4 promotes translation of the chloroplast atpB/E mRNA. Plant J, 2012, 72 [4], 547-558
Zoschke, R.; Kroeger, T.; Belcher, S.; Schöttler, M.A.; Barkan, A.; Schmitz-Linneweber, C.
(See online at https://doi.org/10.1111/j.1365-313X.2012.05081.x) - A rapid ribosome profiling method elucidates chloroplast ribosome behavior in vivo. Plant Cell, 2013, 25 [6], 2265-2275
Zoschke, R.; Watkins, K.P.; Barkan, A.
- Multiple checkpoints for the expression of the chloroplast-encoded splicing factor MatK. Plant Physiol, 2013, 163 [4], 1686-1698
Hertel, S.; Zoschke, R. ; Neumann, L.; Qu, Y.; Axmann, I.M.; Schmitz-Linneweber, C.
(See online at https://doi.org/10.1104/pp.113.227579) - A major role for the plastidencoded RNA polymerase complex in the expression of plastid tRNAs. Plant Physiol, 2014, 164 [1], 239-248
Williams-Carrier, R.; Zoschke, R.; Belcher, S.; Pfalz, J.; Barkan, A.
(See online at https://doi.org/10.1104/pp.113.228726)
