AbstractsBiology & Animal Science

The highly conserved RNA processing exosome appears to provide the major 3’-5’ riboexonucleolytic activity in eukaryotes. In Saccharomyces cerevisiae, the nine subunit core exosome, the nuclear/cytoplasmic riboexonuclease Dis3p, and the nuclear/nucleolar riboexonuclease Rrp6p constitute the nuclear exosome. Despite the critical role of Rrp6p in RNA 3’ end formation and degradation, the dependence of its function on the 9-subunit core exosome and Dis3p remains unclear. The research described here shows that Rrp6p requires one of its two HRDC domains for interaction with the core exosome. In the absence of core interaction, Rrp6p continues to carry out RNA 3’ end processing of 5.8S rRNA and snoRNAs, as well as the degradation of certain truncated rRNA intermediates. Loss of Rrp6p-core exosome interaction results in the inability of the cell to efficiently degrade certain poly(A)+ rRNA processing products known to require the combined activities of Dis3p and Rrp6p. These findings indicate that some of the critical functions of Rrp6p do not require physical interaction with the core exosome. In vivo the nuclear exosome coordinates with the polyadenylating TRAMP complex to hydrolyze aberrant RNAs. Previous reports showed that purified TRAMP enhanced RNA degradation by the nuclear exosome in vitro. However these experiments did not identify the riboexonucleolytic component(s) of the nuclear exosome enhanced by TRAMP. The research described here shows that TRAMP does not significantly enhance RNA degradation by purified core exosomes lacking Rrp6. This suggests that TRAMP activation experiments with nuclear exosome preparations reflect, in part, effects on the activity of Rrp6p. Consistent with this, we show that incubation of purified TRAMP with Gst-Rrp6p results in a ten-fold enhancement in the rate of RNA degradation. This increased activity results from enhancement of the hydrolytic activity of Rrp6p, since TRAMP can not enhance the activity of an Rrp6p variant lacking a key amino acid side chain in its active site. We observed no ATP, or polyadenylation dependence for the enhancement of Rrp6p activity by TRAMP, suggesting that neither the poly(A) polymerase activity of Trf4p nor the helicase activity of Mtr4p play roles in the enhancement. These findings identify Rrp6p as a direct target for TRAMP and indicate that Rrp6p is capable of functioning in core exosome independent RNA degradation pathways. The in vivo and in vitro results presented in this thesis show that Rrp6p activity does not always act in concert with the core exosome complex.