RNA pyrophosphohydrolase (RppH) catalyzes the removal of pyrophosphate from 5' triphosphorylated RNAs thereby initiating RNA degradation. The enzyme has originally been identified in bacteria but homologs are present in eukaryotes where they are thought to be located in plastids or mitochondria. A homolog of the bacterial RNA pyrophosphohydrolase is present in the unicellular green alga Chlamydomonas reinhardtii suggesting that Chlamydomonas RppH has a role in mRNA degradation in the chloroplast of the alga. The purpose of this project was to determine the localization of the RppH homologue in C. reinhardtii. Localization was investigated using two different constructs, a histidine-tagged version of the Chlamydomonas rppH and a histidine-tagged 5’rppH-GFP construct.
A plasmid vector containing Chlamydomonas rppH-6xHN was introduced into C. reinhardtii by nuclear transformation. PCR, RT-PCR, sequencing, and DNA and RNA blotting techniques were used to indentify positive transformants at the DNA and RNA level. In addition, transformants carrying a histidine-tagged 5’rppH-GFP construct, that has previously been transformed and verified to be present at the DNA level, was investigated by RNA blotting. SDS-PAGE, antibodies, mass spectrometry and chloroplast isolation were used to evaluate protein expression from both constructs. In addition a protein activity assay was developed in order to confirm that the Chlamydomonas RppH homolog has RNA pyrophosphohydrolase activity.
Accumulation of RppH-6xHN and 5’RppH-GFP-6xHN proteins in transformants has been detected but the results need to be further substantiated. Localization of RppH was not possible in the time frame of the project because of problems with antibody specificity and with the chloroplast isolation procedure. Further work should focus on analyzing additional transformants and on localization of 5’RppH-GFP-6xHN using confocal microscopy.