The proteins are represented as lines, with conserved regions shown as boxes (CAT, catalytic domain; CD, central domain; RBD, RNA-binding domain; sizes in amino acids are indicated to the right). The green boxes in the sequence alignment mark regions with 50% similarity, light blue boxes indicate 100% similarity, and dark blue and orange boxes are regions with 100% identity. The three conserved catalytic aspartates at positions 236, 238, and 294 are in orange and marked by triangles. Regions with four additional amino acids present in Cid1 and GLD-2 are marked with a orange “4.”

(A) The Trf4 complex has poly(A) polymerase activity. The 5′-end-labeled oligo(A)₁₅ was incubated 30 min with 5, 10, or 20 ng of affinity-purified fractions of the wild-type TAP-tagged Trf4p (Trf4-TAP) or mutant Trf4p with the aspartic acid residues 236 and 238 changed to alanines (DADA-TAP). Protein was omitted in lane 1. Recombinant yeast poly(A) polymerase (Pap1), 1, 2, and 4 ng, was used as a positive control. The migration position of oligo(A)₁₅ is indicated by an arrow.
(B) The Trf4p activity is specific for the addition of adenosine monophosphate. Polyadenylation assays with 20 ng of Trf4-TAP in the presence of different ribonucleoside triphosphates. Recombinant yeast Pap1p, 5 ng, was used as a control. All samples were separated on 15% denaturing gels.

(A) Affinity purification of [His]₆-tagged Trf4 complex identifies five associated polypeptides. Affinity-purified fractions were separated by SDS-PAGE on a 12% gel and stained with Colloidal Coomassie Blue. The protein bands indicated by arrows were identified by MS-MS sequencing. The round holes in the gel lane are a result of punching samples for MS-MS analysis.
(B) Alignment of conserved regions of Air1p and Air2p. Boxes indicate five predicted CCHC-type zinc knuckle motifs. Lines above the sequences mark regions of interaction between Air1p (green) or Air2p (black) with Trf4p, as inferred from a yeast two-hybrid screen.
(C) Polyadenylation activity associated with TAP-tagged versions of proteins identified as components of the Trf4 complex (Trf4, Mtr4, Air1, and Air2) and of Trf5. The 5′-end-labeled oligo(A₁₅) was incubated with 5 or 10 ng of the affinity-purified extracts and analyzed by electrophoresis on a 15% gel. The position of the input RNA is indicated by an arrow. Protein was omitted in lane I. Recombinant yeast poly(A) polymerase (Pap1), 5 or 15 ng, was used as a positive control.
(D) Trf4p and Mtr4p copurified with TAP-tagged proteins identified in the Trf4 complex. Western blot analysis of purified eluates with anti-Trf4p and anti-Mtr4p antibody and antibody against the calmodulin-binding region in the TAP-tag (α-TAP). The same amounts of protein complexes as used in the assay (C) were applied to the gel. The higher molecular weight of the Trf4p-TAP protein is due to the TAP-tag. All antibodies were used at a 1:2,000 dilution.

(A) Polyadenylation assay with Trf4p-TAP and unmodified and native tRNA_i^Met as substrates. The 5′-end-labeled tRNAs were incubated with 50 ng of Trf4 complex for times indicated and resolved by gel electrophoresis. The migration position of the input tRNA is indicated by an arrow.
(B) Sequence of yeast tRNA_i^Met in cloverleaf form with all the known nucleotide modifications. Tertiary interactions are indicated in solid and dashed red lines. The network of hydrogen bonds involving A20, G57, m¹A58, A59, and A60, necessary and unique for tertiary interactions in eukaryotic initiator tRNA, is outlined by solid red lines. A*, 5′-phosphoribosyl-2′-adenosine; D, dihydrouridine; m¹A, 1-methyladenosine; m¹G, 1-methylguanosine; m²G, N²-methylguanosine; m²₂G, N²,N²-dimethylguanosine; m⁷G, 7-methylguanosine; t⁶A, N⁶-threonylcarbamoyladenosine.
(C) Polyadenylation activity of Trf4p-TAP on unmodified wild-type, A34GΔU13 mutant tRNA^Ala, and native tRNA^Ala. The 5′-end-labeled tRNAs were incubated with 20 or 50 ng of Trf4 complex for 30 min at 30 °C. Lane I in each panel shows input tRNA. tRNAs were separated on 10% denaturing gels.
(D) Yeast tRNA^Ala in its cloverleaf form. All known nucleotide modifications are indicated. In the mutant tRNA^Ala used in this study, the uracil at position 13 was deleted, and the A34 in the anticodon loop was changed to guanine (marked in red). ψ, pseudouridine; m¹I³⁷, 1-methylinosine generated by deamination of adenosine at position 37. Other modifications are labeled as in (B).

Polyadenylation assays were performed with radiolabeled unmodified (A and C) and native (B and D) tRNA_i^Met substrate incubated with 25, 50, or 100 ng of Trf4-TAP complex (Trf4-TAP; A and B), or with 5, 10, or 20 ng of recombinant Trf4 protein expressed in the baculovirus system (Trf4-bac; A and B), or with 5, 10, or 20 ng of mutant Trf4-bac (DADA-bac; C and D), or with equal amounts and dilutions of control eluates (CTRL-bac; proteins from cell lysates that unspecifically bound to the Ni²⁺-NTA matrix; C and D). In reconstitution experiments 20 ng of Trf4-bac, or DADA-bac, or control eluates were mixed with 0.5, 3, or 15 ng of recombinant Air1p and/or recombinant Air2p in the combinations indicated. The proteins were pre-incubated for 30 min on ice to allow for binding. Reactions were incubated for 50 min at 30 °C. Control reactions contained no protein (lane 1 in each gel).

(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 Materials and Methods (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.
(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).
(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).

Uncoupled exosome assay on pre-adenylated tRNA. 5′-end-labeled and in vitro polyadenylated unmodified tRNA_i^Met was incubated with 50 ng of affinity-purified Rrp6-TAP eluate alone or in combination with 50 ng of wild-type (Trf4-TAP) or mutant (DADA) complex or 50 ng of Trf4-TAP alone. Reactions were stopped after 30 (lanes 2, 6, 10, and 14), 60 (lanes 3, 7, 11, and 15), 90 (lanes 4, 8, 12, and 16), or 120 min (lanes 5, 9, 13, and 17) and separated on a 15% gel. The arrow indicates the position of the nonadenylated tRNA. Protein was omitted in lane 1. The migration positions of the polyadenylated tRNA (poly[A]) and the degradation products (dp) are indicated by brackets.