| The cellular handling of both coding and noncoding RNAs changes in stress conditions to allow cells to adjust translation of RNAs to different environmental conditions, maintain energy homeostasis, and manage RNA damage. One RNA-binding protein that is involved in stress responses in both prokaryotic and mammalian cells is the Ro protein. Ro contributes to survival after UV irradiation in mammalian cells and in the radiant-resistant eubacterium Deinococcus radiodurans (Chen et al., 2000; Chen et al., 2003). In mammalian cells, the Ro protein accumulates in the nucleus after UV irradiation and in oxidative stress, suggesting that Ro may act in quality control or degradation of nuclear RNAs in certain conditions (Chen et al., 2003; Sim et al., 2009). One hypothesis is that Ro may act with exoribonucleases in stress conditions, because in D. radiodurans, Ro shows genetic interactions with the exoribonuclease polynucleotide phosphorylase (PNP) after UV irradiation and in oxidative stress. However, the function of Ro proteins in stress responses has remained unknown.;In this thesis, I examine the mechanism of Ro involvement in stress conditions by investigating the role of the D. radiodurans Ro ortholog Rsr and exoribonucleases in stress-induced ribosomal RNA processing and degradation. First, I show that efficient 23S rRNA processing, which occurs in heat stress and is known to require Rsr (Chen et al., 2007), also requires three 3'-5' exoribonucleases. Rsr and the nucleases RNase PH, RNase II, and RNase R all likely act in a single pathway for the efficient processing of the 23S rRNA, as strains lacking any of these factors accumulate pre-23S rRNAs with identical 5' and 3' extensions. Additionally, as processing stalls at a stem structure in Rsr- and nuclease-deficient strains, these results support a role for Rsr in promoting nuclease activity through elements of RNA secondary structure.;I also demonstrate the involvement of Rsr in stress-induced rRNA degradation in D. radiodurans. One aspect of the stress response to starvation in bacterial species is the extensive degradation of rRNAs (Jacobson and Gillespie, 1968; Rosset et al., 1966). In prolonged stationary phase in D. radiodurans, this rRNA degradation involves Rsr and PNP. Levels of Rsr and a Rsr-PNP complex increase in this stress condition, and the Rsr-PNP complex fractionates with ribosomal subunits. As PNP fractionation with ribosomal subunits is greatly reduced in the absence of Rsr, I propose that Rsr acts to promote rRNA degradation by increasing the interaction of PNP with rRNA substrates.;Together, these findings support a model wherein Ro proteins function with different nucleases and different RNA substrates in the adaptation of RNA processing and degradation in stress conditions. |
