Polypyrimidine tract binding protein modulates efficiency of polyadenylation

Abstract

Polypyrimidine tract binding protein (PTB) is a major hnRNP protein with multiple roles in mRNA metabolism, including regulation of alternative splicing and internal ribosome entry site-driven translation. We show here that a fourfold overexpression of PTB results in a 75% reduction of mRNA levels produced from transfected gene constructs with different polyadenylation signals (pA signals). This effect is due to the reduced efficiency of mRNA 3' end cleavage, and in vitro analysis reveals that PTB competes with CstF for recognition of the pA signal's pyrimidine-rich downstream sequence element. This may be analogous to its role in alternative splicing, where PTB competes with U2AF for binding to pyrimidine-rich intronic sequences. The pA signal of the C2 complement gene unusually possesses a PTB-dependent upstream sequence, so that knockdown of PTB expression by RNA interference reduces C2 mRNA expression even though PTB overexpression still inhibits polyadenylation. Consequently, we show that PTB can act as a regulator of mRNA expression through both its negative and positive effects on mRNA 3' end processing.

FIG. 1.

Overexpression of PTB reduces steady-state mRNA levels. (A) Gene constructs used to transfect HeLa cells. Each construct is driven by the CMV promoter and uses the β-globin gene as a reporter gene. The α-globin poly(A) signal (α), the β-globin poly(A) signal (β), and the C2 complement poly(A) signal (C2) were placed downstream of the β-globin gene. The β cDNA construct lacks introns. (B) Overexpression of PTB reduces steady-state β mRNA levels up to fivefold. S1 analysis of nuclear and cytoplasmic RNA isolated from HeLa cells transiently transfected with the β construct alone (lanes 1 and 3) or cotransfected with a PTB-expressing plasmid (lanes 2 and 4) followed by denaturing polyacrylamide gel electrophoresis is shown. Lanes 5 (no RNA, no S1 nuclease) and 6 (no RNA) are controls. The VA I adenovirus gene (Va) was used as a cotransfection control (Va cot), confirming equal transfection efficiency and loading of the RNA. Arrows indicate the positions of the pA and Va control bands. Lane M, size markers (in nucleotides). (C) Overexpression of PTB reduces steady-state α mRNA levels up to threefold. S1 analysis of nuclear and cytoplasmic RNA isolated from HeLa cells transiently transfected with the α construct (lanes 1 and 3) or cotransfected with a PTB-expressing plasmid (lanes 2 and 4) is shown. Quantitation of detected β or α signal is shown below gels, with values for transfections without PTB cotransfection set as 1.

FIG. 2.

Reduction in steady-state RNA levels requires PTB expression and is independent of splicing and nascent transcription. (A) Overexpression of PTB in HeLa cells. Western blot analysis of HeLa cell nuclear extract using anti-PTB antibody (diluted 1:2,000) after transfection with a PTB-expressing plasmid (lane 1) or in its absence (lane 2). A control Western blot with actin antibody confirmed equal protein loading (data not shown). (B) Expression of nonsense PTB message has no effect on steady-state RNA levels, while overexpression of PTB reduces the levels threefold. Lane 1, S1 analysis of cytoplasmic RNA isolated from HeLa cells transfected with the β construct; lanes 2 and 3, β cotransfected with a PTB-expressing plasmid (lane 2) or a plasmid carrying a nonsense PTB message (lane 3); lanes 4 and 5, controls (no RNA with or without S1 nuclease treatment), and Va was used as a cotransfection control (Va cot). (C) Reduction in steady-state RNA levels is independent of splicing. S1 analysis of total RNA isolated from HeLa cells transfected with the βcDNA construct (lane 1) is shown. β cDNA cotransfected with plasmids expressing PTB 1 (lane 2), PTB 2 (lane 3), or PTB 4 (lane 4) are shown. Lanes 5 and 6 are controls (no RNA with or without S1), and the Va was used as a cotransfection control. Lane M, size markers; *, 60-nt band. Quantitation for panels B and C is as in Fig. 1. (D) Overexpression of PTB does not affect nascent transcription levels. Nuclear run-on analysis was carried out with β-transfected HeLa cells (β) with β plus cotransfected PTB1 expression plasmid (β+PTB) or with empty pUC18 vector (control). B2, BE2, and B3 are single-stranded M13 probes containing antisense sequences from the β-globin gene as indicated. The histone H4 probe (His) acts as an internal control for Pol II transcription. M13 indicates the probe background. Nylon filters of probe slot blots hybridized to ³²P nuclear RNA were subjected to phosphorimage analysis and show no significant effect of PTB expression on the levels of nascent transcription.

FIG. 3.

Overexpression of PTB reduces the efficiency of mRNA 3′ end processing in vivo. Lane 1, S1 analysis of total RNA isolated from HeLa cells transiently transfected with the β construct; lanes 2 to 4, β cotransfected with PTB1 (lane 2), PTB2 (lane 3), or PTB4 (lane 4); lane 5 (no RNA), negative control. Va was used as a cotransfection control. Arrows indicate the positions of the read-through (RT), pA, and Va cot bands. Quantitation is as for Fig. 1.

FIG. 4.

PTB inhibits cleavage of the α-globin and β-globin poly(A) signals in vitro. In vitro cleavage reactions were performed using SP6 synthetic RNA substrates containing either the β-globin (β-pA) or the α-globin pA signal (α-pA) followed by fractionation using denaturing polyacrylamide gel electrophoresis. The addition of 150 ng of recombinant PTB to the reaction mixture resulted in a threefold inhibition of cleavage (lanes 2). BSA (150 ng) was added to the reaction mixture as a control (lanes 3), or no nuclear extract was added (lanes 4). The position of uncleaved pre-mRNA substrate (input), upstream cleavage products (5′), and downstream cleavage products (3′) are indicated by arrows. Size markers are indicated. In the data quantitation, the proportion of 3′-end-processed RNA for lanes 1 is set as 1.

FIG. 5.

PTB and CstF₆₄ compete for the same pre-mRNA binding sites in a UV cross-linking competition assay. (α-pA) 0, 10, 50, 100, or 250 ng of rPTB was added to a CstF purified protein fraction. Following UV cross-linking, labeled proteins were fractionated by SDS-polyacrylamide gel electrophoresis. As described above, PTB and CstF₆₄ compete for binding to α-globin pA pre-mRNA. (β-pA) 0, 10, 50, 100, 250, or 500 ng of rPTB was added to a CstF purified protein fraction as for α-pA. Again, PTB and CstF₆₄ compete for binding to the β-globin pA pre-mRNA.

FIG. 6.

In vivo knockdown of PTB by RNAi has no significant effect on efficiency of α-globin and β-globin polyadenylation. (A) Western blot analysis of HeLa cells treated with PTB siRNA 1 (lane 1) and siRNA 2 (lane 2), both of which specifically target PTB RNA. NS siRNA is a nonspecific RNAi duplex (lane 3), and in lane 4 no siRNA was transfected into the cells. PTB expression was dramatically reduced, as shown in lanes 1 and 2. Actin was used as an internal control, and its expression remains unchanged (lanes 1 to 4, lower panel). Depletion of PTB in HeLa cells has no effect on the efficiency of the β-globin pA signal (B) or α-globin pA signal (C). S1 analysis of cytoplasmic RNA isolated from HeLa cells transiently transfected with β (B) or α (C) constructs and treated with either siRNA 1 (lanes 1) or siRNA 2 (lanes 2) are analyzed. Cells were treated with an NS siRNA duplex (lanes 3) or with no siRNA (lanes 4). Va was used as a cotransfection control. Lanes M, size markers. Quantitation showed no significant decrease in the levels of steady-state RNA.

FIG. 7.

Both overexpression and in vivo knockdown of PTB by RNAi significantly reduce the C2 complement pA site use. (A) Depletion of PTB in HeLa cells cotransfected with the construct C2 reduces the levels of steady-state RNA up to threefold, while overexpression of PTB also reduces steady-state RNA levels up to threefold. S1 analysis of cytoplasmic RNA isolated from HeLa cells transiently transfected with the C2 construct and treated with either siRNA 1 (lane 1) or siRNA 2 (lane 2) is shown. For lanes 3 and 4, cells were treated with an NS siRNA duplex (lane 3) or with no duplex (lane 4). Va was used as a cotransfection control. Lane M, size markers. On the right panel, an S1 analysis of cytoplasmic RNA isolated from HeLa cells transiently transfected with the C2 construct (lane 1) and cotransfected with a PTB-1-expressing plasmid (lane 2) is shown. Lanes 3 and 4 are controls, minus RNA ± S1 nuclease. Va was used as a cotransfection control (Va cot). (B) PTB and CstF₆₄ compete for the same pre-mRNA binding sites in a UV cross-linking competition assay. Zero, ten, fifty, one hundred, or two hundred fifty nanograms of rPTB was added to a CstF and CPSF purified protein fraction. PTB and CstF₆₄ compete for the C2 complement pA pre-mRNA, as shown by SDS-polyacrylamide gel electrophoresis.