Structure and function of the polymerase core of TRAMP, a RNA surveillance complex

Abstract

The Trf4p/Air2p/Mtr4p polyadenylation (TRAMP) complex recognizes aberrant RNAs in Saccharomyces cerevisiae and targets them for degradation. A TRAMP subcomplex consisting of a noncanonical poly(A) RNA polymerase in the Pol ß superfamily of nucleotidyl transferases, Trf4p, and a zinc knuckle protein, Air2p, mediates initial substrate recognition. Trf4p and related eukaryotic poly(A) and poly(U) polymerases differ from other characterized enzymes in the Pol ß superfamily both in sequence and in the lack of recognizable nucleic acid binding motifs. Here we report, at 2.7-Å resolution, the structure of Trf4p in complex with a fragment of Air2p comprising two zinc knuckle motifs. Trf4p consists of a catalytic and central domain similar in fold to those of other noncanonical Pol β RNA polymerases, and the two zinc knuckle motifs of Air2p interact with the Trf4p central domain. The interaction surface on Trf4p is highly conserved across eukaryotes, providing evidence that the Trf4p/Air2p complex is conserved in higher eukaryotes as well as in yeast and that the TRAMP complex may also function in RNA surveillance in higher eukaryotes. We show that Air2p, and in particular sequences encompassing a zinc knuckle motif near its N terminus, modulate Trf4p activity, and we present data supporting a role for this zinc knuckle in RNA binding. Finally, we show that the RNA 3′ end plays a role in substrate recognition.protein-RNA interactions | RNA quality control T he vast majority of transcripts from eukaryotic genomes do not encode proteins. These transcripts include precursors to noncoding RNAs, such as transfer and ribosomal RNAs, small nuclear and nucleolar RNAs, microRNAs, siRNAs, and piRNAs. These noncoding RNAs often fold into intricate three-dimensional structures that are critical for subsequent processing and for assembling with proteins to form ribonucleoprotein complexes. In addition, genomic sequencing experiments in both yeast and mammals have revealed the existence of a large number of short-lived noncoding transcripts that initiate near RNA polymerase II promoters.

Air1 Zinc Knuckles 4 and 5 and a Conserved IWRXY Motif Are Critical for the Function and Integrity of the Trf4/5-Air1/2-Mtr4 Polyadenylation (TRAMP) RNA Quality Control Complex

“…At present, studies of Air zinc knuckle function have only been carried out in vitro using recombinant protein, and only mutants of ZnK1, ZnK2, and ZnK3 have been generated in the context of Air2 ZnK1-5 (49). Trf4 complexed with an Air2 ZnK1 mutant, but not a ZnK2 or ZnK3 mutant, exhibits impaired polyadenylation of mutant tRNA, suggesting that Air2 ZnK1 is important for RNA recognition (49). An Air2 ZnK4-5 fragment also supports weak Trf4-mediated polyadenylation of aberrant tRNA, indicating that ZnK4 and ZnK5 also have the capacity to recognize RNA (49).…”

“…We also overexpressed trf4 -378 (E378A/E381A in the central domain; see Fig. 8), a nonfunctional mutant of Trf4 containing alanine substitutions of Glu-378 and Glu-381 residues (56), which are close to Air2 residues in the Trf4-Air2 structure and could mediate interactions with the Air proteins (49). As expected, overexpression of Air1 or Air2 completely suppressed the slow growth of air1-C178R cells at 30°C and 37°C (Fig.…”

“…A recent crystal structure of Trf4 in complex with an Air2 fragment shows that Air2 zinc knuckles 4 and 5 interact with the Trf4 central domain (49). In addition, recombinant Trf4 in complex with Air2 ZnK1-5 preferentially polyadenylates aberrant tRNA in vitro, indicating that Air2 zinc knuckles can also recognize RNA (49). A key question is therefore which zinc knuckles of the Air1/2 proteins contribute to RNA recognition and Air protein function in vivo.…”

“…In retroviral nucleocapsid proteins, one or two CX 2 CX 4 HX 4 C zinc knuckles form metalcoordinated reverse turns that typically contact viral singlestranded tetraloop RNA via the variable (C)X 2 and (H)X 4 residues (14,22,47,48). A recent crystal structure of Trf4 in complex with an Air2 fragment shows that Air2 zinc knuckles 4 and 5 interact with the Trf4 central domain (49). In addition, recombinant Trf4 in complex with Air2 ZnK1-5 preferentially polyadenylates aberrant tRNA in vitro, indicating that Air2 zinc knuckles can also recognize RNA (49).…”

“…Trf4 complexed with an Air2 ZnK1 mutant, but not a ZnK2 or ZnK3 mutant, exhibits impaired polyadenylation of mutant tRNA, suggesting that Air2 ZnK1 is important for RNA recognition (49). An Air2 ZnK4-5 fragment also supports weak Trf4-mediated polyadenylation of aberrant tRNA, indicating that ZnK4 and ZnK5 also have the capacity to recognize RNA (49). However, these conclusions are all based on biochemical experiments, and the contributions of the individual zinc knuckles to the in vivo function of the Air proteins have not been addressed.…”