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. 2012 May 8;109(19):7292-7.
doi: 10.1073/pnas.1201085109. Epub 2012 Apr 24.

RNA Unwinding by the Trf4/Air2/Mtr4 Polyadenylation (TRAMP) Complex

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Free PMC article

RNA Unwinding by the Trf4/Air2/Mtr4 Polyadenylation (TRAMP) Complex

Huijue Jia et al. Proc Natl Acad Sci U S A. .
Free PMC article

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.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The unwinding activity of Mtr4p is stimulated in the TRAMP complex. (A) Representative PAGE for unwinding reaction (RNA: 16-bp duplex with 3′ 25-nt single-stranded region, 0.5 nM, 2 mM equimolar dATP-Mg2+) by 200 nM Mtr4p (Left) and 200 nM TRAMP (Right). Aliquots were removed at 1, 3, 10, 20, and 60 min for Mtr4p, and at 0.5, 1, 3, 10, and 20 min for TRAMP. (B) Time courses for unwinding reactions with Mtr4p (○) and TRAMP (●). Conditions were as in A. Data show averages from three independent experiments; error bars represent one SD. Curves represent best fits to the integrated first-order rate law, yielding observed rate constants (kobs). For Mtr4p, kobs, unw = 0.06 ± 0.01 min−1; for TRAMP, kobs, unw = 0.59 ± 0.06 min−1. (C) Dependence of unwinding rate constants (2 mM dATP-Mg2+) on enzyme concentrations for Mtr4p (open circles), WT TRAMP (filled black circles), and TRAMPTrf4-236p (filled gray circles). Rate constants were determined from multiple independent reactions; error bars represent one SD. Curves represent the best fit to the binding isotherm, kunw = kmax, E [E]/([E] + K1/2, E). [E], enzyme concentration. For Mtr4p, kmax, Mtr4p = 0.090 ± 0.003 min−1, K1/2, Mtr4p = 105 ± 36 nM. For WT TRAMP, kmax, TR = 0.84 ± 0.10 min−1, K1/2, TR = 94 ± 31 nM. For TRAMPTrf4-236p, kmax, TR(m) = 0.76 ± 0.06 min−1, K1/2, TR(m) = 105 ± 20 nM. (D) Dependence of unwinding rate constants on ATP and dATP concentrations for Mtr4p (800 nM). Rate constants were determined from multiple reactions; error bars represent one SD. Curves represent the best fit to a binding isotherm, kunw = kmax, dATP [dATP]/([dATP] + K1/2, dATP). With ATP, kmax, ATP = 0.11 ± 0.03 min−1, K1/2, ATP = 1.74 ± 0.75 mM. With dATP, kmax, dATP = 0.11 min−1, K1/2, dATP = 2.08 ± 0.27 mM. (E) Dependence of unwinding rate constants on ATP and dATP concentrations for TRAMPTrf4-236p and WT TRAMP (both at 300 nM). Rate constants were determined from multiple reactions; error bars represent one SD. Curves represent the best fit to the Hill equation kunw = kmax, dATP [dATP]n/([dATP]n + (K1/2, dATP)n). For WT TRAMP, kmax, dATP = 0.56 ± 0.04 min−1, K1/2, dATP = 0.77 ± 0.08 mM, n = 1.9 ± 0.2. For TRAMPTrf4-236p with dATP, kmax, dATP = 0.40 ± 0.01 min−1, K1/2, dATP = 0.56 ± 0.03 mM, n = 3.4 ± 0.7. For TRAMPTrf4-236p with ATP, kmax, ATP = 0.43 ± 0.03 min−1, K1/2, ATP = 0.65 ± 0.08 mM, n = 1.9 ± 0.3.
Fig. 2.
Fig. 2.
Mtr4p and TRAMP unwind a 36-bp RNA duplex. (A) Representative PAGE for unwinding reaction (RNA: 36-bp duplex with 3′ 25-nt single-stranded region, 0.5 nM, 2 mM equimolar dATP-Mg2+) by 200 nM Mtr4p. Aliquots were removed at 1, 3, 10, 20, and 60 min. (B) Representative PAGE for unwinding reaction (RNA and conditions as in A) by 200 nM TRAMP. Aliquots were removed at 0.5, 1, 3, 10, and 20 min. (C) Time course for unwinding of the 36-bp duplex RNA by Mtr4p (conditions as in A). Data show averages from three independent experiments; error bars indicate one SD. For curve fitting, see Fig. S1 (observed unwinding rate constant kobs, unw = 0.02 ± 0.02 min−1). (D) Time course for unwinding of the 36-bp duplex RNA by TRAMP (conditions as in A). Data show averages from three independent experiments; error bars indicate one SD. For curve fitting, see Fig. S1 (kobs, unw = 0.22 ± 0.05 min−1).
Fig. 3.
Fig. 3.
Duplex unwinding by Mtr4p and TRAMP requires an RNA bottom strand. Time courses of unwinding reactions of DNA/RNA hybrid duplexes by Mtr4p (A) and TRAMP (B) (conditions as in Fig. 1). In the 16-bp RNA duplex with the 25-nt single-stranded region (Fig. 1A), top, bottom, or both RNA strands (R) were replaced by DNA (D), as indicated. Duplexes with DNA bottom strands were not unwound. Mtr4p unwound the substrate with the DNA top strand at kobs, unw = 0.07 ± 0.01 min−1; TRAMP at kobs, unw = 0.54 ± 0.07 min−1.
Fig. 4.
Fig. 4.
Simultaneous polyadenylation and unwinding reaction by TRAMP reveals the oligo(A) length required for duplex unwinding. (A) Experimental design for simultaneous polyadenylation and unwinding reaction. A 16-bp duplex with a single unpaired nucleotide at the 3′ terminus was immobilized via a biotin moiety (gray dot; asterisk marks the radiolabel) on streptavidin beads. The Reaction was started by adding TRAMP (300 nM final, 2 mM ATP-Mg2+). After 10 min, the sample was centrifuged, and beads and supernatant were separated and then analyzed on denaturing PAGE. The circled numbers refer to samples and controls analyzed in B. (B) Representative denaturing PAGE (Left) and quantification of unwinding for each RNA species (Right). Lane 1: RNA before reaction; lane 2: RNA after reaction and before centrifugation; lane 3: RNA on beads sample after centrifugation; lane 4: RNA in supernatant after centrifugation. The number of adenines appended (A0 … An) is indicated. The plot (Right) shows the fraction of unwound duplex for individual adenylated species (signals from lane 4 of the PAGE, divided by the sum of signals from lanes 3 and 4 for each species). The sum of signals in lanes 3 and 4 faithfully represents the distribution of species in the unsedimented RNA in lane 2 (Fig. S3). The dashed line indicates A4. (C) Unwinding reactions (10 min, conditions as in Fig. 1) for 16-bp substrates with defined overhangs of three, four, and five adenylates (A3–A5, 4- to 6-nt overhang) corresponding to RNAs in B. The lane marked 95 °C shows heat-denatured duplexes.
Fig. 5.
Fig. 5.
Mtr4p and TRAMP preferably unwind substrates with short single-stranded regions containing adenylates. (A) Unwinding of 16-bp duplexes (Fig. 1) with 4- to 6-nt single-stranded overhangs containing adenosines (●) or a sequence without adenosines (○) by Mtr4p (400 nM; Left) and TRAMP (400 nM; Right). Observed unwinding rate constants (kunw) are averages from three independent measurements (2 mM dATP-Mg2+; other conditions as in Fig. 1); error bars represent the SD. (B) Observed unwinding rate constants for a 16-bp duplex with a 6-nt single-stranded overhang containing adenosines (●) or a sequence without adenosines (○) as a function of Mtr4p (Left) and TRAMP (Right) concentration. Conditions were as in A, observed unwinding rate constants (kunw) are averages from multiple independent measurements; error bars represent the SD. Curves represent best fits to the binding isotherm, kunw = kmax, E [E]/([E] + K1/2, E). [E], enzyme concentration; K1/2, functional affinities; kmax, E, unwinding rate constant at enyzme saturation. Obtained values were as follows: Mtr4p, A-rich substrate; kmax, Mtr4p = 0.66 ± 0.10 min−1; K1/2, Mtr4p = 477 ± 145 nM; Mtr4p, the non-A substrate, kmax, Mtr4p = 0.55 ± 0.21 min−1, K1/2, Mtr4p = 1,697 ± 908 nM; TRAMP, A-rich substrate; kmax, TR = 2.81 ± 0.81 min−1; K1/2, TR = 225 ± 126 nM; TRAMP, non-A substrate; kmax, TR = 1.15 ± 0.10 min−1; K1/2, TR = 657 ± 81 nM. (C) Functional affinities and unwinding rate constants at enzyme saturation for substrates with 6-nt and 25-nt overhangs containing adenosines (A) or a sequence without adenosines (R) for Mtr4p (Left) and TRAMP (Right).
Fig. 6.
Fig. 6.
Coordination between polyadenylation and unwinding activities in TRAMP. ○, number of added adenylates; ●, minimal number of adenylates involved in Mtr4p binding. See text for further explanation.

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