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Structure and Function of the Polymerase Core of TRAMP, a RNA Surveillance Complex

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Structure and Function of the Polymerase Core of TRAMP, a RNA Surveillance Complex Structure and function of the polymerase core of TRAMP, a RNA surveillance complex Stephanie Hamilla, Sandra L. Wolina,b,1, and Karin M. Reinischa,1 aDepartments of Cell Biology, and bMolecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520 Edited* by Stephen C. Harrison, Harvard Medical School, Boston, MA, and approved July 13, 2010 (received for review March 16, 2010) The Trf4p/Air2p/Mtr4p polyadenylation (TRAMP) complex recog- truncated 5S rRNA and signal recognition particle RNAs, aber- nizes aberrant RNAs in Saccharomyces cerevisiae and targets them rant rRNA processing intermediates, and a large class of bidirec- for degradation. A TRAMP subcomplex consisting of a noncanoni- tional transcripts that initiate near RNA polymerase II promoters. cal poly(A) RNA polymerase in the Pol ß superfamily of nucleotidyl The exosome is conserved in higher eukaryotes, and higher transferases, Trf4p, and a zinc knuckle protein, Air2p, mediates eukaryotes have homologs for each component of TRAMP initial substrate recognition. Trf4p and related eukaryotic poly(A) (3, 4), suggesting that the TRAMP-exosome pathway may be and poly(U) polymerases differ from other characterized enzymes widely conserved. TRAMP consists of the poly(A) polymerase in the Pol ß superfamily both in sequence and in the lack of recog- Trf4p or its close homolog Trf5p (65% sequence identity), the zinc nizable nucleic acid binding motifs. Here we report, at 2.7-Å knuckle protein Air2p or its close homolog Air1p (45% sequence resolution, the structure of Trf4p in complex with a fragment of identity), and Mtr4p, a member of the DExH/D box RNA helicase Air2p comprising two zinc knuckle motifs. Trf4p consists of a superfamily 2. The Trf4p/Air2p subcomplex polyadenylates the 3' catalytic and central domain similar in fold to those of other non- ends of aberrant noncoding RNAs, providing a single-stranded canonical Pol β RNA polymerases, and the two zinc knuckle motifs “landing pad” that allows the exosome to initiate RNA decay of Air2p interact with the Trf4p central domain. The interaction (3, 4). surface on Trf4p is highly conserved across eukaryotes, providing A key question in noncoding RNA quality control is how evidence that the Trf4p/Air2p complex is conserved in higher RNAs are recognized as aberrant. In the TRAMP-exosome eukaryotes as well as in yeast and that the TRAMP complex pathway, substrate recognition is mediated by the Trf4p/Air2p may also function in RNA surveillance in higher eukaryotes. We subcomplex (5–8), and in vitro, this subcomplex preferentially show that Air2p, and in particular sequences encompassing a zinc Met polyadenylates an unmodified form of tRNA i over the fully knuckle motif near its N terminus, modulate Trf4p activity, and modified version (6, 9). An understanding of TRAMP substrate we present data supporting a role for this zinc knuckle in RNA recognition will derive from a better understanding of Trf4p/ binding. Finally, we show that the RNA 3′ end plays a role in Air2p and its interactions with RNAs. substrate recognition. Trf4p/5p belongs to a family of ribonucleotide transferases in the Pol ß superfamily of nucleotidyl transferases (10, 11). It con- protein-RNA interactions ∣ RNA quality control sists of N- and C-terminal sequences that have little predicted secondary structure, a catalytic domain similar in sequence to he vast majority of transcripts from eukaryotic genomes do several Pol ß members with known structures, and an adjacent Tnot encode proteins. These transcripts include precursors “central domain,” which shares a short nucleotide recognition to noncoding RNAs, such as transfer and ribosomal RNAs, small motif hx(I/L/V)(E/Q)(E/D/N)PhxxxxNxx (h, hydrophobic; x, nuclear and nucleolar RNAs, microRNAs, siRNAs, and piRNAs. any residue) with so-called noncanonical Pol β RNA polymerases. These noncoding RNAs often fold into intricate three-dimen- Trf4p/5p differs from many structurally characterized ribonucleo- sional structures that are critical for subsequent processing and tidyl transferases in lacking a recognizable RNA binding domain, for assembling with proteins to form ribonucleoprotein com- and it has been proposed that the Air proteins function in RNA plexes. In addition, genomic sequencing experiments in both binding (3, 4). In both Air1p and Air2p, five adjacent CCHC zinc yeast and mammals have revealed the existence of a large number of short-lived noncoding transcripts that initiate near RNA knuckles are inserted between N- and C-terminal sequences pre- polymerase II promoters. Finally, cells contain a variety of long dicted to lack secondary structure. Cx1-2Cx3-6Hx7-10C-type zinc noncoding RNAs, some of which function to regulate gene knuckles (C, cysteine; H, histidine; x, any amino acid) and their expression and chromatin structure (1, 2). interactions with RNAs have been studied in the context of the Because defective RNAs can arise by synthesis from mutant retroviral nucleocapsid proteins (12). In these proteins, residues genes, transcriptional errors, or aberrant processing events, and x1-2 and x8-10 are typically involved in interactions with single- because some products of pervasive genome transcription may stranded, looped regions of RNA, and linker regions between be harmful, cells have evolved surveillance mechanisms to recog- knuckles may bind RNA duplex regions. It has not been estab- nize aberrant and unneeded noncoding RNAs and target them lished, however, whether the Air proteins or their zinc knuckles for degradation by exoribonucleases. Because all exoribonu- are involved in RNA binding, or whether these interactions cleases require a single-stranded end to initiate decay, the initial resemble those of the nucleocapsid zinc knuckles. recognition of target RNAs is often carried out by polymerases that, by adding extra nucleotides to the 3' ends, recruit the decay Author contributions: S.H., S.L.W., and K.M.R. designed research; S.H. performed research; BIOCHEMISTRY machinery. Because many exonucleases are unable to degrade S.H., S.L.W., and K.M.R. analyzed data; and S.H., S.L.W., and K.M.R. wrote the paper. structured RNAs, this machinery frequently includes RNA The authors declare no conflict of interest. helicases (3). *This Direct Submission article had a prearranged editor. One of the best established noncoding RNA degradation path- Data deposition: The atomic coordinates and structure factors have been deposited in the ways in Saccharomyces cerevisiae involves the Trf4p/Air2p/Mtr4p Protein Data Bank, www.pdb.org (PDB ID code 3NYB). polyadenylation (TRAMP) complex and the nuclear exosome, a 1 0 0 To whom correspondence may be addressed. E-mail: [email protected] or Karin. major 3 → 5 exonuclease (3, 4). In vivo, TRAMP and the exo- [email protected]. some are involved in the degradation of a large variety of RNA This article contains supporting information online at www.pnas.org/lookup/suppl/ substrates, including hypomodified and unspliced pre-tRNAs, doi:10.1073/pnas.1003505107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1003505107 PNAS ∣ August 24, 2010 ∣ vol. 107 ∣ no. 34 ∣ 15045–15050 Downloaded by guest on September 30, 2021 Here we investigate the architecture and function of the Trf4p/ A ZK1-5 ZK4-5 Air2p heterodimer. We show that Air2p and its most N-terminal WT tRNA mutant tRNA WT tRNA mutant tRNA time (min) 0 1 2.5 5 10 20 0 1 2.5 5 10 20 0 1 2.5 5 10 20 0 1 2.5 5 10 20 zinc knuckle are important for the polyadenylation activity of 900, 1114 Trf4p, and show data suggesting that this zinc knuckle interacts 692 489,501 with tRNA substrates. We have also determined, at a resolution 320 of 2.7 Å, the structure of a Trf4p/Air2p subcomplex consisting of 242 the Trf4p catalytic and central domains and a segment of Air2p 190 that includes the fourth and fifth zinc knuckles. The fold of Trf4p is similar to that of other noncanonical Pol β family enzymes de- 147 spite insignificant sequence similarity in the central domain. The 124 structure shows that the fourth and fifth zinc knuckles of Air2p 110 interact with the central domain of Trf4p. The Trf4p surface that MW they bind is highly conserved in a wide range of eukaryotes, im- (bases) portant experimental data supporting conservation of the Trf4p/ Air2p complex in higher eukaryotes. lane 1 3 5 7 9 11 13 15 17 19 21 23 Results and Discussion B 1.0 Characterization of Trf4p/Air2p Core Complexes. Due to significant WT tRNA + ZK1-5 sample degradation during purification (Fig. S1), we were not able 0.8 to isolate a complex consisting of full-length forms of Trf4p and mutant tRNA + ZK1-5 Air2p. We therefore worked with Trf4p/Air2p subcomplexes, 0.6 WT tRNA + ZK4-5 where the proteolytically sensitive N and C termini of both mutant tRNA + ZK4-5 4 ∕ 2 0.4 proteins were removed. One subcomplex, Trf p Air pZK1-5, con- sisted of the catalytic and central domains of Trf4p (residues 161– 0.2 481) and the five zinc knuckles of Air2p (residues 58–198); a 4 ∕ 2 second version, Trf p Air pZK4-5, included only the fourth and polyadeylated fraction 0.0 fifth zinc knuckles of Air2p (residues 119–198). We coexpressed 0 5 10 15 20 Trf4p and Air2p constructs in Escherichia coli, and purified time (min) subcomplexes by affinity chromatography and gel filtration. D The truncated complexes were tested for their polyadenylation C activity and also for the ability to distinguish between aberrant and 0.8 correct forms of tRNAs. In these experiments, we used wild-type tRNAAla and a mutant that was identified as a preferred substrate 0.6 for the intact TRAMP complex purified from yeast (6). Both trun- 0.4 cated forms of Trf4p/Air2p are active in polyadenylation assays (Fig.
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