The Polyadenylation Activity of the Trf4 Complex Stimulates the Degradation of Unmodified tRNA_i^Met by the Nuclear Exosome

Abstract

<div><p>(A) The PAP activity of Trf4 complex is required to stimulate the exosome activity. In a coupled exosome/polyadenylation assay, 5′-end-labeled unmodified tRNA_i^Met was incubated with 50 ng of affinity-purified Rrp6-TAP eluate for 30 min as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030189#s4" target="_blank">Materials and Methods</a> (lane 2), followed by addition of 50 ng of wild-type (Trf4-TAP), mutant complex (DADA-TAP), or buffer A (buffer). Reactions were stopped after 10 (lanes 3, 7, and 11), 30 (lanes 4, 8, and 12), 60 (lanes 5, 9, and 13), or 90 min (lanes 6, 10, and 14) and separated on a 15% gel. Arrows indicate the position of the input tRNA. Protein was omitted in lane 1 of each gel. The migration positions of the degradation products (dp) are indicated by a bracket.</p> <p>(B) Coupled polyadenylation/exosome assay. The 5′-end-labeled unmodified tRNA_i^Met was pre-adenylated with 50 ng of affinity-purified Trf4-TAP complex for 30 min (lane 2). Then 50 ng of exosome complex (Rrp6-TAP) or buffer A (buffer) was added, and the reactions were continued as in (A).</p> <p>(C) Depletion of Mtr4p results in incomplete degradation. Coupled-assay, 5′-end-labeled unmodified tRNA_i^Met was pre-incubated for 30 min with Trf4p-TAP lacking Mtr4p (Trf4-TAP w/o Mtr4), followed by the addition of 50 ng of Rrp6-TAP complex or buffer A, and the incubation was continued as in (A).</p></div