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In vascular plants and eukaryotic algae, chloroplasts are the sites for photosynthesis that converts light energy into stored chemical food that benefits both plants and animals. Photosynthesis in chloroplasts requires proteins that are encoded in both chloroplasts genes and nuclear genes, and the coordination of expression of all these genes is important for this process. Many molecular events are involved in expression and regulating the chloroplast-encoded genes, and we study aspects of RNA stability and translation which are some of these key events. Previous work has shown that a 5'-3' exoribonucleolytic activity exists in the chloroplasts of Chlamydomonas reinhardtii and is involved in degrading chloroplasts mRNAs that encode photosynthetic chloroplast proteins. Such RNA degradation in chloroplasts may be catalyzed by an 5'-3' exoribonucleases (Xrn)-like enzymes or possibly an RNaseJ1-like enzyme. Genomic evidence indicates that C. reinhardtii has three nuclear Xrn genes, described here as CrXrn1, 2 and 3.
Currently, we are primarily focusing on the CrXrn1 gene, its expression and function. RT-PCR and sequencing data indicate that the CrXrn1 gene, with homologous sequence to the yeast Xrn1, generates two distinct mRNAs (CrXrn1a and CrXrn1b) via alternative splicing of an exon near the 5' end of the coding region. The CrXrn1b mRNA has a second in-frame translation start site that results in the addition of 40 extra amino acids at the N-terminus of the CrXrn1b protein. This N-terminal extension has sequence and structural similarities to C. reinhardtii chloroplast targeting sequences. To assess sub-cellular locations, we are developing reporter genes to express CrXrn-GFP fusion proteins to test the cellular location (nucleus, cytosol, chloroplast, or mitochondria?) of CrXrn1a and CrXrn1b proteins, the latter may be targeted to the chloroplast where it could regulate chloroplast mRNA stability.
As an alternative method to test sub-cellular location, we are also using cell fractionation and immunoblotting methods to assess location of CrXrn1 proteins. To do this, an anti-CrXrn1 antibody was generated using a synthetic peptide and preliminary evidence indicates that this cross-reacts with an approximately 200-kDa protein, the expected size of the CrXrn1 protein, in both whole cell proteins extracts and proteins from purified chloroplasts.
To test biochemical, we have expressed both CrXrn1a and CrXrn1b in E. coli and tested for 5'-3' exonuclease activity using an in vitro assay and analysis of degradation products by both Thin Layer Chromatography (TLC) and polyacrylamide gels. Both CrXrn1a and CrXrn1b have demonstrated RNase activity that is consistent with a 5'-3' exonucleases.
A second topic of research in the lab is our continued investigation of the small regulatory sequences ("elements") in the Chlamydomonas chloroplast petD mRNA 5' UTR and the function of these RNA elements. Using site-directed mutagenesis and chloroplast transformation, we are investigating the role of the element I petD mRNA sequence located at the very 5' end. We are also using a bioinformatics approach to try to identify new RNA regulatory elements in chloroplast 5' UTRs. For this, a computer algorithm that uses an "oligo-search method" is being combined with our microarray data on non-photosynthetic mcd1 mutants using a Self-Organizing Tree Algorithm (SOTA) to correlate expression with conserved small RNA sequences. A putative conserved sequences has been identified with this method and may function for RNA stability, translation or both in a subset of chloroplast genes. This bioinformatic approach complements the molecular biology research in the lab.
Current lab members:
- Michael Fischer, MAMB graduate student
- Ashley Gehrand, MAMB graduate student
- Jacob Tatay, MAMB graduate student
Former lab members:
- Brian Erickson, MAMB graduate student
- Theresa Dailey, Biological Sciences undergraduate
- José Henriquez, Biological Sciences undergraduate
- Rachel Jarrad, REU summer student
- Jamie Jaskolski, Biological Sciences undergraduate
- Nathaniel Jeanson, Molecular Biology and Bioinformatics undergraduate
- Lynn Kramzar, Molecular Biology undergraduate and MAMB graduate student
- Jennifer Lavendear, Molecular Biology and Bioinformatics undergraduate
- Angela Leis, Molecular Biology undergraduate
- Ed Manteufel, Biological Sciences undergraduate
- Toby Mueller, MAMB graduate student
- Aaron Naatz, REU summer student
- Dane Popovski, Biological Sciences undergraduate
- Kate Schassberger, Biological Sciences undergraduate
- Kyle Upchurch, MAMB graduate student
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Questions, comments or suggestions should be e-mailed to David Higgs (higgs@uwp.edu)
