TRAMP complex enhances RNA degradation by the nuclear exosome component Rrp6.
The RNA-processing exosome contains ribonucleases that degrade aberrant RNAs in archael and eukaryotic cells. In Saccharomyces cerevisiae, the nuclear/nucleolar 3'-5' exoribonuclease Rrp6 distinguishes the nuclear exosome from the cytoplasmic exosome. In vivo, the TRAMP complex enhances the ability of the nuclear exosome to destroy some aberrant RNAs. Previous reports showed that purified TRAMP enhanced RNA degradation by the nuclear exosome in vitro. However, the exoribonucleolytic component(s) of the nuclear exosome enhanced by TRAMP remain unidentified. We show that TRAMP does not significantly enhance RNA degradation by purified exosomes lacking Rrp6 in vitro, suggesting that TRAMP activation experiments with nuclear exosome preparations reflect, in part, effects on the activity of Rrp6. Consistent with this, we show that incubation of purified TRAMP with recombinant Rrp6 results in a 10-fold enhancement of the rate of RNA degradation. This increased activity results from enhancement of the hydrolytic activity of Rrp6 because TRAMP cannot enhance the activity of an Rrp6 mutant lacking a key amino acid side chain in its active site. We observed no ATP or polyadenylation dependence for the enhancement of Rrp6 activity by TRAMP, suggesting that neither the poly(A) polymerase activity of Trf4 nor the helicase activity of Mtr4 plays a role in the enhancement. These findings identify TRAMP as an exosome-independent enhancer of Rrp6 activity.
Callahan
Kevin P
KP
From the Departments of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642.
Butler
J Scott
JS
From the Departments of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642; Departments of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642; the Center for RNA Biology: From Genome to Medicine, University of Rochester Medical Center, Rochester, New York 14642. Electronic address: scott_butler@urmc.rochester.edu.
D000255:Adenosine Triphosphate / Q000378:metabolism; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005095:Exoribonucleases / Q000235:genetics / Q000378:metabolism*; D063326:Exosome Multienzyme Ribonuclease Complex; D055354:Exosomes / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism; D026723:Polyadenylation; D012331:RNA, Fungal / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D011061:Poly A; D000255:Adenosine Triphosphate; D004259:DNA-Directed DNA Polymerase; C487545:PAP2 protein, S cerevisiae; D005095:Exoribonucleases; D063326:Exosome Multienzyme Ribonuclease Complex; C542283:DIS3 protein, S cerevisiae; C112610:RRP6 protein, S cerevisiae; C101004:MTR4 protein, S cerevisiae; D053487:DEAD-box RNA Helicases