Predominant action of Nab2p versus Pab1p in poly(A) tail length control. Polyadenylation kinetics on the pre-cleaved CYC1 precursor (CYC1pre). Two 12-fold reactions were set up and kinetics were initiated by addition of radiolabeled CYC1pre. A 20 µl reaction contains: 30 µg of pab1Δ (YAS394) 3′-end-processing competent yeast cell extract and either 500 ng of recombinant Pab1p (lanes 1–9) or a combination of 500 ng of recombinant Pab1p with 440 ng of recombinant Nab2p (lanes 10–18). Twenty microliters from each reaction mixture were withdrawn at the indicated time points, and the RNA content was extracted, separated on a 6% polyacrylamide-8.3 M urea denaturing gel and visualized by autoradiography. M, molecular weight markers were as described in Figure 1.

Polyadenylation of mature polyadenylated CYC1 and 3′-end accessibility assays. (A) Polyadenylation reactions of gel-purified polyadenylated CYC1pA were performed in vitro with TAP-purified CPF, 40 ng recombinant CF IB/Nab4p, and increasing amounts of recombinant Nab2p (lanes 3–5) or Pab1p (lanes 9–11). Reactions were further supplemented with the same amount of TAP-purified CPF after 1 h and the reactions were pursued for an additional hour (lanes 6 and 12). 3′-end accessibility was tested after 1 h of reaction by addition of 24 ng of yeast poly(A) polymerase Pap1p to reactions containing the highest concentrations of Nab2p (lanes 7 and 8) or Pab1p (lane 13). Reactions were further incubated for another hour. ‘++’ denotes addition of a 1.5 molar excess of poly(A) polymerase compared to Nab2p instead of 24 ng. Lane 1, unreacted precursor. (B) Polyadenylation reactions with CYC1pre RNA (lane 1, unreacted precursor) were performed with the TAP-purified 3′-end-processing machinery [CF IA+CPF] with 40 ng recombinant CF IB/Nab4p and 500 ng of either recombinant Nab2p (lanes 3 and 4) or Pab1p (lanes 5 and 6). Accessibility to further elongation was challenged after 1 h reaction as in A. The solid black bar highlights the increase of hyperpolyadenylated species upon addition of recombinant Pap1p to the Pab1p-containing reaction. (C) Densitometric analysis of the gel in B using ImageJ. Black arrows are highlighting the significant amount of hyperpolyadenylated species present in Pab1p-containing reactions in comparison with Nab2p-containing reactions. TOP and BOTTOM designate corresponding positions on the gel in B. Lane numbers of the gel in B are indicated on the left side of each corresponding densitogram. Reaction products were analyzed and visualized as in Figure 1. M, molecular weight markers were as described in Figure 1.

Poly(A) tail length control activity of various Nab2p deletion mutants. (A) Schematic representation of various Nab2p deletion mutants. The numbers in brackets indicate the extent of the corresponding deletion. Nter, amino terminal part of the protein. (Q)₁₁, poly-glutamine stretch comprising residues 106 to 116. (QQQP)₉, nine successive repeats of the tetrapeptide Gln-Gln-Gln-Pro comprising residues 121 to 156. RGG, arginine/glycine rich region of the protein comprising residues 201 to 264. (Cx₅Cx_4-6Cx₃H)₇, seven repeats of the indicated zinc-finger domain sub-type spanning residues 262 to 473. (B) Cleavage and polyadenylation reactions were reconstituted in vitro as described in Materials and Methods with the CYC1 precursor (lanes 1, 12, 20, 28, 35, 48), the TAP-purified 3′-end formation machinery [CF IA+CPF], recombinant CF IB/Nab4p, and either Nab2p or the indicated mutant protein as polyadenylation regulation factor. − or + indicate reactions in the absence or in the presence of 420 nM of wild-type Nab2p (500 ng in 20 µl), respectively; ++ indicates a reaction with 1.6 µM of Nab2p (2 µg in 20 µl). Triangles represent increasing concentrations of the indicated mutant protein. In the cases of nab2-21, nab2ΔQrich, svNab2, NterQrich, the concentrations used were: 84, 168, 250 and 420. For GST-RGG-(CCCH)₇, nab2Δ(CCCH)₇, (CCCH)₇, the concentrations were: 84, 168, 250, 334 and 420 nM. For RGG-(CCCH)₇, the concentrations were 26, 52, 78, 104 and 130 (lanes 51–55) and 260, 390, 520 and 650 nM (lanes 56–59). Reaction products were analyzed and visualized as in Figure 1. M, molecular weight markers were as described in Figure 1. Lanes 28–34 were on the same gel, and lanes 35–47 as well.

Topology analysis of Nab2p/poly(A), nab2ΔRGGp/poly(A) and Pab1p/poly(A) complexes. (A) Poly(A)₂₀₀ alone (lanes 1–5) or poly(A)₂₀₀/protein complexes (lanes 6–17) were digested with micrococcal nuclease (NS7) at the indicated concentrations as described in Materials and Methods. RNA fragments were extracted, ethanol precipitated and subjected to electrophoresis on a 12% polyacrylamide-8.3 M urea gel. Visualization was done by autoradiography. The black arrow points to the weak signal of the 73-nt long species. (B) Densitometric analysis of the gel shown in A using ImageJ. TOP and BOTTOM indicate corresponding positions on the gel as in A. The black arrow is as in A.

Effect of the simultaneous presence of Nab2p and Pab1p on polyadenylation regulation in a reconstituted reaction in vitro. (A) Cleavage and polyadenylation reactions were reconstituted in vitro as described in Materials and Methods with the CYC1 radiolabeled precursor (lane 1), TAP-purified machinery [CF IA+CPF] and recombinant Nab4p/Hrp1p/CF IB. Poly(A) tail length control was assayed by addition of recombinant Pab1p in combination with recombinant Nab2p either as concomitant (lanes 3–7) or relative (lanes 8–17) titrations. (B) Cleavage and polyadenylation reactions were reconstituted as in A, but poly(A) tail length control was assayed by addition of recombinant Pab1p only (lanes 3–7). (C) Quantification of hyperpolyadenylation occurring in the indicated lanes of gels in A and B. The abundance of polyadenylated species with sizes higher than the 309 nt precursor was quantified with ImageJ and expressed as percentage of the maximum hyperpolyadenylation that takes place in the absence of any poly(A)-binding proteins lanes 2 of A and B. Gels and corresponding lane numbers are specified along with their respective value. Reaction products were analyzed on a 6% polyacrylamide-8.3 M urea denaturing gel and visualized by phosphorimaging. M, labeled MspI-digested pBR322 was used as a molecular weight marker and corresponding sizes are indicated in number of nucleotides. Upstream and downstream cleavage products are indicated by grey and white rectangles respectively. AAAA represents the poly(A) tail.

Cleavage inhibition of mature polyadenylated CYC1pA by Pab1p or Nab2p. (A) Cleavage assays with gel-purified mature polyadenylated CYC1pA were done in an in vitro reconstituted reaction using the TAP-purified 3′-end-processing machinery [CF IA+CPF] and 40 ng of recombinant Nab4p when specified. Inhibition of cleavage was challenged by increasing amounts of either Nab2p (lanes 4–9) or Pab1p (lanes 12–16). The major cleavage site is indicated by a number 1, and the alternative cryptic cleavage sites are numbered 2 and 3, as previously described (24). Lanes 1–9 were on the same gel. Lane 1, unreacted precursor. (B) Cleavage reactions with the CYC1 precursor (lanes 1 and 7) were performed as in A, except that 40 ng of CF IB/Nab4p were included in all assays. Inhibition of cleavage was tested by adding increasing amounts of either Nab2p (lanes 2–6) or Pab1p (lanes 8–11). Lanes 7–11 were on the same gel. Lane 10 contained a spillover of lane 9 and was therefore skipped. Lane 1, unreacted precursor. Reaction products were analyzed and visualized as in Figure 1. M, molecular weight markers were as described in Figure 1.

Nab2p and Pab1p inhibit poly(A) polymerase Pap1p with similar efficiencies. Polyadenylation reactions with 5′-end radiolabeled poly(A)₃₄ precursor were done with 24 ng of recombinant poly(A) polymerase Pap1p in a final volume of 20 µl. Pap1p activity was challenged by addition of increasing amounts of either Nab2p (lanes 3–8) or Pab1p (lanes 9–14) recombinant proteins. Reaction products were analyzed on an 8% polyacrylamide-8.3 M urea denaturing gel and visualized by autoradiography. Lane 1, unreacted precursor. M, molecular weight markers were as described.

Functional analysis of the RGG-box region of Nab2p. (A) Cleavage and polyadenylation reactions were reconstituted in vitro as described in Materials and Methods with the CYC1 precursor, the TAP-purified 3′-end formation machinery [CF IA+CPF], recombinant CF IB/Nab4p, and increasing amounts of either Nab2p (lanes 2–6) or nab2ΔRGG mutant protein (lanes 7–10) as polyadenylation regulation factors. Lane 1, unreacted precursor. (B) Polyadenylation reactions of the gel-purified polyadenylated CYC1pA were performed in vitro with TAP-purified CPF, 40 ng of recombinant CF IB/Nab4p, and increasing amounts of recombinant nab2ΔRGG mutant protein (lanes 3–5). Reactions were further supplemented with the same amount of TAP-purified CPF after 1 h (lane 6) as in Figure 4A. 3′-end accessibility was challenged by addition poly(A) polymerase to the reaction (lane 7) as in Figure 4A. Reaction products were analyzed and visualized as in Figure 1. Lane 1, unreacted precursor. M, molecular weight markers were as described in Figure 1. (C) RNA 3′-end accessibility of Nab2p/poly(A)₆₀ or nab2ΔRGG/poly(A)₆₀ complexes was challenged by titrations of recombinant poly(A) polymerase Pap1p. Five hundred nanograms of Nab2p or nab2ΔRGGp were pre-incubated with 3 nM of poly(A)₆₀ for 15 min at 30°C, and polyadenylation reactions were initiated by addition of the indicated Pap1p amounts and further incubated for 1 h. RNA contents were then extracted, separated on a 8% polyacrylamide-8.3 M urea denaturing gel and visualized by phosphorimaging. Lane 1: poly(A)₆₀ alone. M, molecular weight markers were as described in Figure 1. (D) Polyadenylation efficiency of poly(A)₆₀ complexed to either Nab2p or nab2ΔRGGp was calculated from experiments such as in C. The abundance of polyadenylated species with sizes higher than the 60 nt precursor was quantified with ImageJ, normalized to the integrated density in each entire lane and expressed as a ratio to the abundance of polyadenylated species higher than the 60 nt precursor in the absence of Pap1p (as in C, lane 2). Values for 5, 10, 24, 42 and 960 ng of Pap1p are means from three independent experiments ± standard error. Values for 100, 300 and 500 ng of Pap1p are means from two independent experiments ± standard error.