Molecular Genetic Analysis of 5’-3’ Exoribonuclease (Xrn) Mutants in the Model Green Alga Chlamydomonas reinhardtii

Daniela Ysabel Castellanos, Valerie E Riehl, Megan M. Agustine, Aaron B. Maldonado

Abstract


Organisms regulate the expression of genes that can help control development and responses to environmental changes.  In the case of plants and algae, nucleus-encoded regulatory proteins help control photosynthesis in chloroplasts, and some of these regulatory proteins control the stability of chloroplast mRNAs that in turn alter protein abundance.  Candidate nuclear genes predicted to regulate chloroplast gene expression are being investigated in the single-celled alga Chlamydomonas reinhardtii, a model organism used to study photosynthesis.  Sequence analysis predicts that C. reinhardtii has four 5’-3’ exoribonuclease (Xrn) genes.  Two appear to encode Type 1 Xrn proteins, typically targeted to the cytoplasm, while the other two appear to encode Type 2 Xrn proteins, typically targeted to the nucleus.  To investigate the function of these predicted exoribonucleases we have obtained ten C. reinhardtii mutant strains, each with a predicted DNA insertion into one of the Xrn genes.  The mutants are from the recently reported Indexed, Mapped Mutant Library (CLiP) in C. reinhardtii.  Our initial growth tests confirmed that all ten mutants contain the selectable paromomycin resistance gene AphVIII, and we are using an initial PCR screen to test location of the inserted DNA within the Xrn genes.  Those mutants that contain the predicted insertion within a targeted Xrn gene will be further analyzed for changes in growth phenotypes compared to a wild-type control, while grown under standard conditions.  The long-term goal is to assess molecular phenotypes to identify changes in abundance or processing of candidate RNAs and their effects.  Ultimately, RNA-Seq (transcriptomic) analysis would be used to test mutants for genome-wide changes in RNA abundance and sequence.  Complete analysis of these mutants will provide a better understanding of the function of Xrn’s in algae and possible impacts on photosynthesis.

Keywords


Chlamydomonas; 5'-3' exoribonuclease; RNA stability

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