RNA unwinding by the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex

Abstract

Many RNA-processing events in the cell nucleus involve the Trf4/ Air2/Mtr4 polyadenylation (TRAMP) complex, which contains the poly(A) polymerase Trf4p, the Zn-knuckle protein Air2p, and the RNA helicase Mtr4p. TRAMP polyadenylates RNAs designated for processing by the nuclear exosome. In addition, TRAMP functions as an exosome cofactor during RNA degradation, and it has been speculated that this role involves disruption of RNA secondary structure. However, it is unknown whether TRAMP displays RNA unwinding activity. It is also not clear how unwinding would be coordinated with polyadenylation and the function of the RNA helicase Mtr4p in modulating poly(A) addition. Here, we show that TRAMP robustly unwinds RNA duplexes. The unwinding activity of Mtr4p is significantly stimulated by Trf4p/Air2p, but the stimulation of Mtr4p does not depend on ongoing polyadenylation. Nonetheless, polyadenylation enables TRAMP to unwind RNA substrates that it otherwise cannot separate. Moreover, TRAMP displays optimal unwinding activity on substrates with a minimal Mtr4p binding site comprised of adenylates. Our results suggest a model for coordination between unwinding and polyadenylation activities by TRAMP that reveals remarkable synergy between helicase and poly(A) polymerase.he Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex is involved in nuclear RNA surveillance, 3′-end processing of rRNA, small nucleolar RNAs (snoRNAs), and snRNAs, and in gene silencing and chromatin maintenance (1-3). The TRAMP complex consists of three subunits that are highly conserved in eukaryotes: a noncanonical poly(A) polymerase (Trf4p or Trf5p in Saccharomyces cerevisiae), a Zn-knuckle protein (Air2p or Air1p), and the RNA helicase Mtr4p (4-7). TRAMP assists RNA degradation by the nuclear exosome, most notably by appending short (∼4-5 nt) oligo(A) tails at the 3′ ends of RNAs slated for exosome-mediated degradation (4,5,(8)(9)(10). In addition, it has been speculated that TRAMP enables the nuclear exosome to efficiently degrade structured RNA through unwinding activity associated with Mtr4p (1, 2, 11-13).However, RNA helicase activity has not been demonstrated for TRAMP. Though Mtr4p alone has been shown to unwind RNA duplexes in vitro (12, 13), it is not known whether binding of Trf4p/Air2p abolishes, decreases, increases, or otherwise alters this helicase activity. This question is important for TRAMP function, because Mtr4p and Trf4p operate with opposite polarities. Mtr4p only unwinds duplexes with a 3′ unpaired region, i.e., with a 3′ to 5′ polarity (12, 13). Trf4p polyadenylates the 3′ end of RNA and thus possesses 5′ to 3′ polarity (4, 5). How unwinding and polyadenylation with opposite polarities are coordinated in one complex is not readily apparent.Moreover, Mtr4p controls the lengths of poly(A) tails appended by TRAMP (9). This role of Mtr4p requires ATP, but does not involve unwinding (9). Instead, Mtr4p binds to the 3′ end of the RNA, detects the number of 3′-terminal adenosines, and, in response, modulates ATP affinity and adenylatio...

The association of a La module with the PABP-interacting motif PAM2 is a recurrent evolutionary process that led to the neofunctionalization of La-related proteins

“…Whether AtLARP6a may play a role in the noncoding RNA TRAMPmediated surveillance system is not straightforward to predict on the basis of current knowledge. The oligo(A) tails appended at the 39 ends of the RNAs to be degraded by this process are probably too short (3-5 residues) (Jia et al 2011(Jia et al , 2012 for the optimal interaction with AtLARP6a, but since this La module shows some affinity to other homopolymers (Fig. 7), we cannot rule out that it could bind to internal sequences as well as to the short oligo(A) tail of TRAMP substrates.…”

Ski2-like RNA helicase structures

“…107 Similarly, Mtr4 helicase activity is enhanced simply by binding to Trf4-Air2. 45 In the context of TRAMP, Mtr4 has the ability to detect the length of a poly(A) tail, which directly influences Trf4 and Mtr4 activity. 45,107 A point mutation in the ratchet domain (E947A) disrupts modulation of Trf4 activity by as a trimer, 86 but subsequent mass spec analysis indicates that it is a tetramer containing two copies of Ski8.…”

“…45 In the context of TRAMP, Mtr4 has the ability to detect the length of a poly(A) tail, which directly influences Trf4 and Mtr4 activity. 45,107 A point mutation in the ratchet domain (E947A) disrupts modulation of Trf4 activity by as a trimer, 86 but subsequent mass spec analysis indicates that it is a tetramer containing two copies of Ski8. 87 Activation of the exosome also requires an accessory protein, Ski7, that has been shown to physically link the two protein machineries.…”

“…Whether Mtr4 is directly reading the sequence in this context is unclear, although Mtr4 does exhibit an affinity for polyadenylated RNAs over nonpolyadenylated substrates in in vitro binding and unwinding assays. 45,109,110 Understanding the assembly of the TRAMP complex is an active focus of investigation. Trf4 and Air2 are tightly associated in vitro, remaining in complex above 1M NaCl, while Mtr4 interaction is more dynamic and dissociates from Trf4-Air2 around 500 mM NaCl.…”

“…TRAMP adds a short (~5-nt) poly(A) tail to the 3' end of RNA substrates, 107,108 which is preferentially unwound by Mtr4 45 and subsequently degraded by the exosome. Remarkably, Mtr4 directly modulates the polyadenylation activity of Trf4.…”