Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Gustavo C. Macintosh
T2 RNases have been identified in numerous organisms from plants to animals and even microorganisms. The distribution of this family in almost every organism suggests it may have an important biological function that has being conserved through evolution. In plants, two different subfamilies are defined. S-RNases are involved in pollen rejection during self-incompatible interactions, while S-like RNases are a more diverse group, with not clear function.
While expression studies suggest that S-like RNases are involved in many stress responses, including defense against pests and nutrient starvation, and in developmental processes such as senescence, functional studies addressing their biological role are still lacking. In an attempt to fill this gap in knowledge we initiated an analysis of RNS1, a RNase T2 enzyme from Arabidopsis thaliana . We showed that RNS1 transcript and protein are induced during mechanical wounding of the plant and by treatment with the hormone Abscisic Acid (ABA). We found that ABA is part of the RNS1 wounding response pathway; yet in the absence of ABA the RNS1 transcript is still induced. Thus, RNS1 defines a novel wound-response pathway, independent of known wounding signals such as oligogalacturonides, jasmonates, and ethylene. The unusual regulation of RNS1 by novel ABA-dependent and ABA-independent wounding response pathways suggest a unique, yet undefined, function.
To further study the function of T2 RNases, we extended our work to other organisms. We found that petunia nectar is rich in RNase activities, and we identified four T2 RNases in Petunia hybrida . Two of these RNases are similar to S-like RNases; while the other two contain characteristics similar to both S- and S-like RNases. The latter two (RNase Phy3 and RNase Phy4 ) also show patterns of regulation consistent with those of nectarins; suggesting they may have a role in petunia nectar defense.
While expression analyses can provide clues to understand function of RNases, it was clear that the neither of these potential defense roles would be the one selected to keep this family in almost all organisms. Thus, we carried out phylogenetic analyses in search of conservation patterns that could provide more information about this elusive biological role. To this end we characterized RNase T2 proteins from animals (zebrafish) and plants (rice) and identified RNase T2 genes from a variety of species with fully sequenced genomes. We identified two T2 RNase genes in the Danio rerio (zebrafish) genome. Patterns of regulation for these RNases suggest a possible housekeeping function. Evolutionary analysis of these enzymes along with the emergence of the RNase A family suggest many of the "stress" related functions preformed by T2 RNases in plants are carried out by the RNase A family in vertebrates; yet retention of at least one T2 RNase suggests an essential function exists.
Expression analysis of eight T2 RNases from Oryza sativa (rice) and phylogenetic analysis of plant T2 RNases present in other fully sequenced plant genomes to led us to conclude that plant S-like RNases are divided in two classes; with RNases in Class I showing signs of rapid evolution and a possible function in stress responses (defense, nutrient deficiency), whereas Class II RNases are expressed ubiquitously and phylogenic conservation suggests a possible housekeeping role. This housekeeping role may be conserved for RNase T2 proteins in animals, while Class I functions are carried out by RNase A proteins in vertebrates.
Melissa Sue Hillwig
Hillwig, Melissa Sue, "Regulation, function, and evolution of T2 RNases" (2009). Graduate Theses and Dissertations. 11095.