A New Yeast Poly(A) Polymerase Complex Involved in RNA Quality Control

Abstract

Eukaryotic cells contain several unconventional poly(A) polymerases in addition to the canonical enzymes responsible for the synthesis of poly(A) tails of nuclear messenger RNA precursors. The yeast protein Trf4p has been implicated in a quality control pathway that leads to the polyadenylation and subsequent exosome-mediated degradation of hypomethylated initiator tRNAMet (tRNAi Met). Here we show that Trf4p is the catalytic subunit of a new poly(A) polymerase complex that contains Air1p or Air2p as potential RNA-binding subunits, as well as the putative RNA helicase Mtr4p. Comparison of native tRNAi Met with its in vitro transcribed unmodified counterpart revealed that the unmodified RNA was preferentially polyadenylated by affinity-purified Trf4 complex from yeast, as well as by complexes reconstituted from recombinant components. These results and additional experiments with other tRNA substrates suggested that the Trf4 complex can discriminate between native tRNAs and molecules that are incorrectly folded. Moreover, the polyadenylation activity of the Trf4 complex stimulated the degradation of unmodified tRNAi Met by nuclear exosome fractions in vitro. Degradation was most efficient when coupled to the polyadenylation activity of the Trf4 complex, indicating that the poly(A) tails serve as signals for the recruitment of the exosome. This polyadenylation-mediated RNA surveillance resembles the role of polyadenylation in bacterial RNA turnover.

Execution of the meiotic noncoding RNA expression program and the onset of gametogenesis in yeast require the conserved exosome subunit Rrp6

“…We next examined the poly-A polymerase Pap2, a component of the TRAMP complex, which enhances Rrp6's activity (23,24). Although Pap2 was suggested to act in the same pathway as Rrp6 (9), an RT-PCR analysis of MUTs in MATa/α pap2 cells revealed no mitotic accumulation and showed their meiotic expression to be indistinguishable from the one observed in wild-type strains (Fig.…”

The nuclear poly(A) polymerase and Exosome cofactor Trf5 is recruited cotranscriptionally to nucleolar surveillance

“…These at least include the targeting of misassembled prerRNAs, recognized by as yet unknown mechanisms, for polyadenylation by the nuclear poly(A) polymerase TRAMP complexes and selective 39 / 59 exoribonucleolytic digestion by the Exosome (Dez et al 2006(Dez et al , 2007 for review, see . The TRAMP complexes are comprised of a zinc knuckle and putative RNA binding protein (either Air1 or Air2), a DExD/H-box family helicase (Mtr4/Dob1), and a poly(A) polymerase (either Trf4 or Trf5), the later defining the TRAMP4 or TRAMP5 complexes, respectively (LaCava et al 2005;Vanacova et al 2005;Wyers et al 2005; for reviews, see Andersen et al 2008). The Exosome consists of 10 core subunits of which Rrp44/Dis3 is directly active in RNA synthesis and degradation, carrying both exoRNase and endoRNase activities (Dziembowski et al 2007;Schneider et al 2007;.…”

Addition of poly(A) and poly(A)-rich tails during RNA degradation in the cytoplasm of human cells

“…It has recently become clear that almost the entire genome is transcribed, which results in the production of antisense and noncoding RNAs (13-17). These cryptic unstable transcripts were found to undergo rapid poly(A)-assisted decay as well (10,11,14). However, although such nuclear mechanisms have been reported, poly(A)-assisted RNA decay has not been found in the cytoplasm of eukaryote cells.…”

“…In yeast nuclei, transient poly(A) was found to play a role in RNA quality control wherein polyadenylation, initiated by the TRAMP complex, targets incorrectly folded tRNA molecules to degradation by the nuclear exosome (10,11). In human cells, cotranscriptionally cleaved 3′ regions of an introduced β-globin gene and a class of short, highly unstable RNAs, dubbed PROMPTs (promoter upstream transcripts), were found to be adenylated and accumulated when the exosome was downregulated (12,13).…”