Frederick J. LaRiviere

A Convergence of rRNA and mRNA Quality Control Pathways Revealed by Mechanistic Analysis of Nonfunctional rRNA Decay

Eukaryotes possess numerous quality control systems that monitor both the synthesis of RNA and the integrity of the finished products. We previously demonstrated that Saccharomyces cerevisiae possesses a quality control mechanism, nonfunctional rRNA decay (NRD), capable of detecting and eliminating translationally defective rRNAs. Here we show that NRD can be divided into two mechanistically distinct pathways: one that eliminates rRNAs with deleterious mutations in the decoding site (18S NRD) and one that eliminates rRNAs containing deleterious mutations in the peptidyl transferase center (25S NRD). 18S NRD is dependent on translation elongation and utilizes the same proteins as those participating in no-go mRNA decay (NGD). In cells that accumulate 18S NRD and NGD decay intermediates, both RNA types can be seen in P-bodies. We propose that 18S NRD and NGD are different observable outcomes of the same initiating event: a ribosome stalled inappropriately at a sense codon during translation elongation.

Chapter 12. Analysis of nonfunctional ribosomal RNA decay in Saccharomyces cerevisiae.

Mature rRNA are normally extremely stable in rapidly growing cells. However, studies show that some mature rRNA in Saccharomyces cerevisiae are, in fact, turned over quite rapidly by the nonfunctional rRNA decay (NRD) pathway. NRD eliminates the RNA component of mature but defective ribosomal subunits and ribosomes. NRD was discovered using rDNA reporter plasmids to express and track the fate of rRNA containing mutations in functionally important regions of the ribosome. This chapter outlines some of the available rDNA reporter plasmids that can be used to study NRD and describes assays to test for functionality and stability of rRNA in yeast.

Quantitative PCR analysis of diepoxybutane and epihalohydrin damage to nuclear versus mitochondrial DNA

The bifunctional alkylating agents diepoxybutane (DEB) and epichlorohydrin (ECH) are linked to the elevated incidence of certain cancers among workers in the synthetic polymer industry. Both compounds form interstrand cross-links within duplex DNA, an activity suggested to contribute to their cytotoxicity. To assess the DNA targeting of these compounds in vivo, we assayed for damage within chicken erythro-progenitor cells at three different sites: one within mitochondrial DNA, one within expressed nuclear DNA, and one within unexpressed nuclear DNA. We determined the degree of damage at each site via a quantitative polymerase chain reaction, which compares amplification of control, untreated DNA to that from cells exposed to the agent in question. We found that ECH and the related compound epibromohydrin preferentially target nuclear DNA relative to mitochondrial DNA, whereas DEB reacts similarly with the two genomes. Decreased reactivity of the mitochondrial genome could contribute to the reduced apoptotic potential of ECH relative to DEB. Additionally, formation of lesions by all agents occurred at comparable levels for unexpressed and expressed nuclear loci, suggesting that alkylation is unaffected by the degree of chromatin condensation.