• Biochemical Characterization of the Essential Activities of Saccharomyces cerevisiae Mtr4p

      Bernstein, Jade; Toth, Eric A.; Hassel, Bret A. (2010)
      Accurate processing of precursor RNA and timely degradation of aberrant RNAs is crucial for proper cell function. A variety of RNAs are initially synthesized as long precursors, which must then be trimmed to form functional RNAs. Any byproducts of this trimming as well as any defective RNAs must be rapidly degraded. These processing events are mediated in part by RNA degradation machinery consisting of an exonuclease complex called the exosome and the helicase Mtr4p. Mtr4p is a critical partner of the exosome that presumably maintains the momentum of exonucleolytic decay/processing by removing structural impediments from the target RNAs. Our studies have examined the RNA binding parameters of Mtr4p showing that Mtr4p binds single stranded RNA in a length and nucleotide-dependent manner. These studies also showed that Mtr4p has a unique interaction with poly(A) RNA substrates. The interaction between Mtr4p and poly(A) RNA may facilitate targeting of polyadenylated RNAs to the exosome. We have investigated the mechanism underlying the preference of Mtr4p for poly(A) substrates as a means to understand how Mtr4p might facilitate targeting. Our analysis has revealed that Mtr4p interacts with poly(A) via a mechanism that is distinct from the mechanism used when it interacts with other substrates. In addition, we show that homopolymeric stretches like poly(A) suppress the ATPase activity of Mtr4p. Suppression of activity correlates with a decrease in the rate of complex dissociation. These findings indicate that the Mtr4p-poly(A) complex is unique and ideally suited for targeting to the exosome. Taken together, these studies offer characterization of some of the essential activities of Saccharomyces cerevisiae Mtr4p and provide insight into how it might function within the context of the nuclear exosome.