Localization of a putative RNA pyrophosphohydrolase in the unicellular green alga Chlamydomonas reinhardtii

Abstract

In bacteria an RNA pyrophosphohydrolase (RppH) has been shown to remove pyrophosphate (PPi) from the 5’ end of mRNAs thereby allowing binding of RNases that degrade the transcripts. In previous master projects a gene that coded for a protein with RppH activity was found in the Chlamydomonas nuclear genome suggesting a function of this protein in this alga similar to the function in mRNA breakdown in bacteria. The goal of this project was to localize the RNA pyrophosphohydrolase (RppH) in cells of Chlamydomonas reinhardtii. It was hypothesized that the Chlamydomonas RppH has a function in compartments that harbor a prokaryotic machinery for mRNA degradation, i.e. chloroplasts and mitochondria, since cytoplasmic mRNAs, which lack triphosphates at their 5’ ends, are not substrates for this enzyme. In silico analysis of the Chlamydomonas RppH sequence identified a putative chloroplast transit peptide of 33 amino acids at its N-terminus. In this project the putative transit peptide was fused to the N-terminus of two fluorescent proteins, mCherry Cr and tdTomato, that have previously been used in localization studies in Chlamydomonas. A recombinant gene construct, consisting of a gene that confers resistance to the antibiotic zeocin and the fluorescent protein gene fused to the nucleotide sequence of the putative transit peptide, was transformed into Chlamydomonas and expression of the construct analyzed by PCR and fluorescence microscopy. Despite being resistant to zeocin a fluorescent protein gene could only be detected in a small number of the transformants analyzed. Similarly, fluorescence microscopy detected only a few cells among many that expressed a fluorescent protein. In those cells localization of the fluorescent protein in the chloroplast correlated with the presence of a transit peptide in the construct used for transformation. It is concluded that the putative transit peptide targets RppH to the chloroplast of Chlamydomonas. This conclusion, though, needs to be substantiated by analyses of more transformants.