Polyadenylation of SV40 late pre-mRNA is dependent on phosphorylation of an essential component associated with the 3' end processing machinery

Abstract

We investigated whether phosphorylation of the essential components involved in the 3' end processing of mRNAs was required for mRNA polyadenylation. The proteins in HeLa nuclear extract were dephosphorylated with alkaline phosphatase, which is known to remove the phosphate moieties from serine and tyrosine. The dephosphorylated extract was used for analyzing cleavage-dependent polyadenylation of SV40 late pre-mRNA. The phosphatase treatment of the extract completely blocked the polyadenylation reaction, whereas dephosphorylation of the extract did not inhibit the cleavage reaction. Since the cleavage depends upon functional integrity of the specificity factor, it is unlikely that the phosphorylated state of the latter factor is required for the 3' end processing. Sodium vanadate, a potent inhibitor of alkaline phosphatase, markedly reduced the inhibitory effect of the phosphatase on the polyadenylation reaction. Dephosphorylation of the extract also prevented formation of the polyadenylation-specific complex with pre-mRNA, whereas the cleavage-specific complexes were formed under this condition. The Mn-dependent polyadenylation, which is largely poly(A) extension reaction, was relatively resistant to the phosphatase treatment. These data indicate that phosphorylation of a key factor is essential for the 3' end processing of pre-mRNA, and suggest that the factor may be poly(A) polymerase.

Figure 1

Inhibition of polyadenylation by dephosphorylation of components in the HeLa nuclear extract. HeLa nuclear extract was incubated with increasing amounts of calf intestinal alkaline phosphate (0.01 U/μg to 0.08 U/μg) for 30 minutes at 30°C. The samples were then assayed for 3′ end processing using a full-length SV40 mRNA substrate under Mg²⁺ conditions. One sample (lane 1) consisted of untreated HeLa extract in the presence of 3′ dATP and Mg²⁺ to detect the upstream cleavage product. Arrows a, b, and c correspond to the polyadenylated RNA, −58/+55 SV40 late pre-mRNA (containing the upstream and downstream recognition sequences and the cleavage site), and the 5′ cleavage product, respectively. Lanes 2 to 8 correspond to samples incubated with 0, 0.015, 0.02, 0.03, 0.04, 0.06, and 0.08 U/μg of alkaline phosphatase, respectively.

Figure 2

Effect of alkaline phosphatase on the cleavage of SV40 L mRNA. HeLa nuclear extract was incubated with calf intestinal alkaline phosphatase, as in Figure 1. The samples were then assayed for cleavage in the presence of 3′ dATP and Mg²⁺ and varying amounts of alkaline phosphatase. Lanes 1–7 correspond to 0.01, 0.015, 0.02, 0.03, 0.04, 0.06, and 0.08 U/μg of alkaline phosphatase, respectively. Control samples consisted of incubation of the samples under the following conditions: lane 8, incubation for 30 minutes at 30°; lane 9, l0mM Hepes pH 7.6 in a volume to match that of the 0.08 U/μg; lane 10, 0.08 U/μg of alkaline phosphatase that was previously boiled for 5′; lane 11, a sample processed under normal conditions Without alkaline phosphatase.

Figure 3

Effect of sodium vanadate on the inhibitory effect of alkaline phosphatase. Sodium vanadate, a potent inhibitor of alkaline phosphatase, was preincubated with alkaline phosphatase at concentrations previously shown to be inhibitory for polyadenylation. HeLa nuclear extract was then reacted with the pretreated phosphatase and further processed as described in the legend to Figure 1. Lanes 5–7 correspond to samples incubated with 0.01,0.015, and 0.02 U/μg of alkaline phosphatase, respectively, and lanes 2–4 represent samples incubated with corresponding concentrations of pretreated phosphatase. Inhibition of alkaline phosphatase by vanadate negated the inhibitory effects of dephosphorylation on polyadenylation compared to the sample incubated with untreated phosphatase (compare lane 4 with 7). Note that at the lower concentration of the pretreated phosphatase, the 5′ cleavage product was not detected, in contrast to the sample incubated with untreated phosphatase (compare lane 2 with lane 5).

Figure 4

Effect of alkaline phosphatase on the Mn²⁺ activated cleavage-independent polyadenylation. HeLa nuclear extract was incubated with varying amounts of alkaline phosphatase, as previously described. The samples were then processed for polyadenylation of SV40 L pre-mRNA in the presence of Mg²⁺ (lanes 1–6) or Mn²⁺ (lanes 7–12). Lanes 2 to 5 contained 0.01,0.02,0.04, and 0.06 units/μg of alkaline phosphatase, respectively. Lanes 8 to 11 contained similar units of the phosphatase. Lanes 1 and 7 represent untreated samples, whereas lanes 6 and 12 represent samples incubated with Hepes buffer in a volume equal to that of the highest concentration of the phosphatase used.

Figure 5

Effect of dephosphorylation on the formation of RNP complexes under polyadenylation conditions. HeLa nuclear extract was dephosphorylated with alkaline phosphatase (0.04 U/μg). Control samples consisted of extract incubated with 10 mM Hepes pH 7.6 in a volume equal to that of treated samples. Polyadenylation reactions were carried out for varying time intervals ranging from 0 to 90 minutes using [32P]-labeled SV40 pre-mRNA substrate. Half of the reaction (12.5 μl) was terminated by the addition of heparin to a final concentration of 5 mg/ml and incubated for an additional 10 minutes at 30°C. These samples were analyzed on a 4% non-denaturing polyacrylamide gel at 4°C (A). The remaining samples were deproteinized and analyzed on an 8% acrylamide, 8.3 M Urea gel (B). A. Lanes 2 to 5 correspond to the dephosphorylated samples incubated for 0, 5, 30, and 90 minutes respectively, while lanes 6 to 9 correspond to the untreated sample incubated for the same time periods. Electrophoresis on the native gel showed that in the primary stages of the RNP complex formation, where cleavage takes place, the dephosphorylated and non-dephosphorylated samples formed comparable complexes with pre-mRNA (compare lanes 2 and 3 with lanes 6 and 7). However, as the reaction progressed from cleavage to polyadenylation, the treated samples were unable to form a final polyadenylated RNP complex (compare lanes 4 and 5 with lanes 8 and 9). The arrow on the left corresponds to pre-mRNA used as the substrate. B. Electrophoresis of the remaining half of the samples under denaturing conditions showed the state of the RNA corresponding to the complexes formed in A. The emergence of the polyadenylated RNA correlated with the formation of the polyadenylation-specific complexes (P); compare B, lanes 7 and 8 with A, lanes 8 and 9. The sample corresponding to A, lane 1 was not processed under denaturing conditions, as it represented only the labeled pre-mRNA probe.

Figure 6

Effect of dephosphorylation on the formation of RNP complexes under cleavage conditions. HeLa nuclear extract was dephosphorylated with 0.04 U/μg of alkaline phosphatase. Control samples were incubated with 10 mM Hepes pH 7.6 in a volume equal to that of the treated samples. Cleavage reactions were carried out for varying time intervals ranging from 0 to 30 minutes. The reactions were terminated by the addition of heparin to a final concentration of 5 mg/ml and incubated for an additional 10 minutes at 30°C. These samples were analyzed on a 4% non-denaturing poly-acrylamide gel. Lanes 2 to 4 correspond to the dephosphorylated samples incubated for 0, 5, and 30 minutes, respectively, whereas lanes 5 to 7 correspond to the untreated samples incubated for the same time intervals. The native gel showed that the formation of the pre-cleavage complex (B) and the post-cleavage complex (B′) by the dephosphorylated samples was comparable to that formed by the untreated samples (compare lane 2 with lane 5, 3 with 6, and 4 with 7).