Poly(A) tail length is controlled by the nuclear poly(A)-binding protein regulating the interaction between poly(A) polymerase and the cleavage and polyadenylation specificity factor

Abstract

Poly(A) tails of mRNAs are synthesized in the cell nucleus with a defined length, approximately 250 nucleotides in mammalian cells. The same type of length control is seen in an in vitro polyadenylation system reconstituted from three proteins: poly(A) polymerase, cleavage and polyadenylation specificity factor (CPSF), and the nuclear poly(A)-binding protein (PABPN1). CPSF, binding the polyadenylation signal AAUAAA, and PABPN1, binding the growing poly(A) tail, cooperatively stimulate poly(A) polymerase such that a complete poly(A) tail is synthesized in one processive event, which terminates at a length of approximately 250 nucleotides. We report that PABPN1 is required to restrict CPSF binding to the AAUAAA sequence and to permit the stimulation of poly(A) polymerase by AAUAAA-bound CPSF to be maintained throughout the elongation reaction. The stimulation by CPSF is disrupted when the poly(A) tail has reached a length of approximately 250 nucleotides, and this terminates processive elongation. PABPN1 measures the length of the tail and is responsible for disrupting the CPSF-poly(A) polymerase interaction.

FIGURE 1.

Stimulation of poly(A) polymerase by CPSF bound to straight poly(A) is sensitive to PABPN1. A, CPSF can stimulate poly(A) polymerase when bound to straight poly(A). 80 fmol of L3preA₁₅ or A₁₀₀ was incubated in a 25-μl reaction with an approximately equimolar amount of CPSF, 20 fmol of PAP and 1.6 pmol of His-PABPN1 as indicated. After 2 min of preincubation at 37 °C, the reactions were started by the addition of ATP. The reactions were stopped after 30 s or 15 min as indicated, and RNAs were analyzed by gel electrophoresis. The size of DNA markers in nucleotides is indicated on the left. B, PABPN1 inhibits the stimulatory activity of CPSF on straight poly(A). 80 fmol of A₂₈₀ was incubated in 20-μl reactions with an approximately equimolar amount of CPSF, 32 fmol of PAP, and increasing amounts of PABPN1 as indicated. 1.5 pmol of PABPN1 was sufficient for A₂₈₀ coverage as judged by an independent gel mobility shift assay. The reactions were preincubated for 10 or 15 min at 37 °C and started by addition of ATP. They were stopped after 15 min and analyzed by gel electrophoresis. A control reaction containing a 10-fold amount of poly(A) polymerase was included as explained under “Experimental Procedures.”

FIGURE 2.

PABPN1 maintains the AAUAA dependence of CPSF stimulation. The reactions were set up containing 80 fmol of RNA/25 μl of unit volume, either wild type (wt) or with a point mutation in the polyadenylation signal (Δ) and with different poly(A) tail lengths, as indicated. The amounts of CPSF were approximately equimolar to the RNA, 20 fmol of poly(A) polymerase, and 1.6 pmol PABPN1 was used as indicated. The reaction mixtures were prewarmed for 3 min at 37 °C, the samples were withdrawn for the 0 min time points, and the reactions were started by the addition of ATP. Aliquots were taken at the time points indicated, and RNA was analyzed by gel electrophoresis. The sizes of DNA markers are indicated on both sides.

FIGURE 3.

CPSF stimulation is sensitive to the distance between AAUAAA and the 3′ end. The stimulation of poly(A) polymerase by CPSF was measured with two sets of RNA substrates with an increasing distance between the CPSF-binding site AAUAAA and the 3′ end. In each case, an identical set of RNAs with a point mutation in AAUAAA was used as a control. Polyadenylation reactions contained, per unit volume of 25 μl, 80 fmol of RNA, and 80 fmol of PAP in the presence or absence of CPSF (amount approximately equimolar with RNA). After warming to 37 °C, aliquots for the 0-min time point were withdrawn, the reactions were started by the addition of ATP, and additional aliquots were withdrawn after 2 and 10 min. A control reaction containing a 10-fold higher amount of poly(A) polymerase (800 fmol) was incubated for 10 min in the absence of CPSF. PAP stimulation was analyzed as described under “Experimental Procedures” based on the 10-min time points. The plotted data were averaged from at least two experiments for each set of RNAs. Raw data for the first series of RNAs and the mutant controls are shown in supplemental Fig. S1. A, distance-dependent stimulation with the first set of RNA substrates having different 3′ end sequences. The black squares represent the data for RNAs with a wild-type AAUAAA sequence and show a decreasing elongation efficiency in proportion to an increasing distance between AAUAAA sequence and 3′ end. Open squares represent the control data obtained with the corresponding RNAs having a point mutation in the AAUAAA sequence. Elongation is less efficient than with the wild-type sequence and essentially independent of the length of the RNA. B, distance-dependent stimulation with the second set of RNA substrates having uniform 3′ end sequences (-UGUA). The data for the wild-type and mutant RNAs are shown as in A.

FIGURE 4.

CPSF and PABPN1 can bind simultaneously to an RNA with a long poly(A) tail. 80 fmol of L3pre-A₃₀₀ orL3preΔ-A₃₀₀ was incubated in a 20-μl reaction volume in the absence of ATP with 100 fmol of PAP, 2400 fmol of calf thymus PABPN1 (saturating as judged by titration in an independent gel mobility shift assay) and CPSF (approximately equimolar with RNA) in the presence of 1.5 μg of competitor tRNA. After 30 min of incubation at room temperature, complexes were resolved on a native polyacrylamide/agarose composite gel. The reactions containing all three proteins were carried out twice (lanes 7 and 8 and lanes 12 and 13, respectively).

FIGURE 5.

PABPN1 inhibits poly(A) polymerase stimulation by CPSF on long tails. Polyadenylation reactions were set up containing, per unit volume of 20 μl, 80 fmol of RNA, 32 fmol poly(A) polymerase and, where indicated, CPSF at a concentration approximately equimolar with RNA and/or 3.5 pmol of PABPN1 wild-type or LALA mutant. RNA, poly(A) polymerase, and PABPN1 were assembled on ice, and the mixture was prewarmed for 10 min at 37 °C. Next CPSF was added, followed by a second preincubation for 15 min at 37 °C. 20-μl aliquots were withdrawn for the 0-min time points, and then polyadenylation was started by the addition of ATP. Additional aliquots were withdrawn after 1, 3, and 5 min for L3pre-A₁₅ (small light gray wedges, lanes 1–24) and after 15, 30, and 60 min for L3pre-A₃₃₀ (large dark gray wedges, lanes 25–48). RNAs were resolved by polyacrylamide gel electrophoresis. The sizes of DNA markers in nucleotides are indicated on the left.

FIGURE 6.

Tail length dependence of the inhibitory effect of PABPN1. The reactions of 20 μl were assembled containing, as indicated, 80 fmol of L3pre-based RNAs with various poly(A) tail lengths, 32 fmol of PAP, CPSF (approximately equimolar with RNA), and increasing amounts of PABPN1 LALA. For each RNA a control reaction containing a 10-fold amount of poly(A) polymerase was also carried out. After addition of RNA, poly(A) polymerase and PABPN1 the reaction mixtures were prewarmed for 10 min at 37 °C. After CPSF addition, prewarming was continued for 15 min, and then polyadenylation was started by ATP addition. The reactions were stopped after 15 min at 37 °C, and the RNA was analyzed by gel electrophoresis. The amount of PABPN1 LALA used corresponds to a calculated 1.3- and 2-fold saturation on L3preA₂₈₀ and -A₃₀₀, a 5- and 8-fold saturation on L3preA₁₁₀, and a 38- and 57-fold saturation on L3pre-A₁₅ as judged by gel mobility shift analyses.

FIGURE 7.

PABPN1 has to coat long poly(A) tails to inhibit CPSF stimulation of poly(A) polymerase. A, titration of the inhibitory effect of PABPN1. 80 fmol of radioactive L3pre-A₃₀₅ was incubated in a 20-μl reaction volume with 1.25 μg of competitor tRNA, 10 fmol of PAP, CPSF approximately equimolar to the RNA, and increasing amounts of PABPN1 LALA as indicated. After 5 min of preincubation at 37 °C, polyadenylation was started by ATP addition and continued for 60 min at 37 °C. Some of the reactions were carried out twice (lanes 5 and 15 and lanes 4 and 16). A control reaction with a 10-fold amount of poly(A) polymerase was also carried out as explained under “Experimental Procedures.” RNA was analyzed by gel electrophoresis. The sizes of DNA markers are indicated on both sides. B, titration of PABPN1 LALA in a gel mobility shift assay. 80 fmol of radioactive L3pre-A₃₀₅, 1.5 μg of tRNA, and increasing amounts of PABPN1 LALA were incubated in 20-μl reaction volumes under polyadenylation conditions except that ATP was left out. After 30 min of incubation at room temperature, complexes were resolved on a native polyacrylamide/agarose composite gel and detected by phosphorimaging.

FIGURE 8.

Only poly(A) is counted as part of the tail: elongation of mixed tails. A, reaction mixtures were assembled on ice containing, per 25-μl unit volume, 80 fmol of RNA, 1.25 μg of tRNA, 10 fmol of PAP, 1200 fmol of PABPN1, and an amount of CPSF approximately equimolar with RNA. Control reactions with a 10-fold amount of PAP were done in parallel. The mixtures were preincubated for 2 min at 37 °C, and the reactions were started by the addition of ATP. Aliquots were withdrawn at the time points indicated. The reaction products were analyzed by gel electrophoresis. Unreacted substrate RNA is shown in the first lane of each of the three sets of reactions. The size of DNA markers (in nucleotides) are indicated on the right. B, line traces showing the RNA size distributions from polyadenylation reactions in A at the 5-min time points. The vertical gray line to the left of the 404-nt marker indicates the peak of the product lengths obtained by extension of L3pre-N₄₉-A₁₅ and L3pre-A₁₅-N₄₉-A₁₅.

FIGURE 9.

Model of length control mechanism. CPSF binds the polyadenylation signal AAUAAA and recruits PAP. The first PABPN1 molecule joins the complex once the oligo(A) tail has reached a length of about 12 nucleotides. Additional PABPN1 molecules cover the growing tail. Formation of a tight, spherical PABPN1 particle on the growing poly(A) tail facilitates folding back of the RNA, which is required to maintain a contact between CPSF and poly(A) polymerase. Thus, the enzyme is held in the complex by cooperative interactions with both CPSF and PABPN1 and can synthesize the entire poly(A) tail in a processive manner. When the poly(A) tail exceeds a critical length of about 250 adenylate residues, additional PABPN1 molecules can no longer be accommodated in the spherical RNA-protein complex, and the contact between poly(A) polymerase and CPSF cannot be maintained. Thus, during further elongation of the poly(A) tail, poly(A) polymerase is held in the complex only by PABPN1; elongation becomes poorly processive and therefore slow.