U2AF modulates poly(A) length control by the poly(A)-limiting element

Abstract

The poly(A)-limiting element (PLE) restricts the length of the poly(A) tail to <20 nt when present in the terminal exon of a pre-mRNA. We previously identified a 65 kDa protein that could be cross-linked to a functional PLE, but not to an inactive mutant element. This binding was competed by poly(U) and poly(C), but not poly(A) or poly(G). Selectivity for the pyrimidine-rich portion of the PLE was demonstrated by RNase footprinting of the binding activity in total nuclear extract. A 65 kDa protein that selectively cross-linked to the functional PLE was purified by conventional chromatography and identified as the large subunit of U2 snRNP auxiliary factor (U2AF). Overexpression of U2AF65 in cells transfected with a PLE-containing reporter construct resulted in the appearance of a population of mRNAs with heterogeneous poly(A) tails. However, this effect was lost following deletion of the C-terminal RNA recognition motifs (RRMs). A C-->G mutation following the AG dinucleotide in the PLE resulted in mRNA with poly(A) ranging from 25-50 nt. This reverted to a discrete, <20 nt poly(A) tail in cells expressing U2AF65. Our results suggest that U2AF modulates the function of the PLE, perhaps by facilitating the binding of another protein to the element.

Figure 1

RNase footprinting of protein binding to PLE RNA. 5′-[³²P]PLE B RNA was incubated on ice with or without HeLa nuclear extract as above, followed by digestion at 37°C with RNase A (lanes 3 and 4) or RNase T1 (lanes 5 and 6) for 15 and 30 s, respectively. The products were separated on a 12% polyacrylamide/6 M urea gel and protected nucleotides were identified relative to the mobility of fragments generated by NaOH treatment of the input RNA (lane 2).

Figure 2

Identification of the 65 kDa PLEBP by EMSA. (A) 5′-[³²P]PLE B RNA was incubated for 30 min on ice without (lane 1) or with nuclear extract (lanes 2–6). The indicated amounts of unlabeled PLE B RNA competitor were added followed by another 30 min incubation. Protein–RNA complexes were then separated on a non-denaturing polyacrylamide gel, which was dried and visualized by autoradiography. (B) Protein–RNA complexes were prepared as in (A) with the addition of a UV cross-linking step prior to gel electrophoresis. The three retarded bands were excised from the dried gel and recovered protein–RNA complexes were digested with RNase A and separated on a 10% SDS–PAGE gel. Lane 1 contains a control of nuclear extract cross-linked to 5′-[³²P]PLE B RNA and lane 5 contains a control of nuclear extract cross-linked to a uniformly ³²P-labeled transcript for the 5′ 160 nt of albumin mRNA.

Figure 3

Homopolymer competition of PLE binding by the 65 kDa PLEBP. 5′-[³²P]PLE B RNA was incubated on ice with HeLa nuclear extract for 30 min, followed by addition of buffer (lanes 1 and 12) or a 10- or 100-fold excess of unlabeled PLE B RNA (lanes 2–3), poly(A) (lanes 4–5), poly(G) (lanes 6–7), poly(C) (lanes 8–9) or poly(U) (lanes 10–11). UV cross-linking and SDS–PAGE was performed after an additional 30 min incubation.

Figure 4

Chromatographic fractionation of the 65 kDa PLEBP. (A) The scheme for fractionation of PLEBP from HeLa nuclear extract is shown. For each column a portion of every fraction was analyzed by UV cross- linking to 5′-[³²P]PLE B RNA and by silver stained SDS–PAGE to assay for binding activity and degree of purification. (B) The selective recovery of PLEBP at each step in the fractionation is shown by UV cross-linking to either the 23 nt wild-type 5′-[³²P]PLE B RNA (PLE B) or a 23 nt 5′-³²P- labeled RNA for the inactive MutG element (MutG). The 65 kDa PLEBP is indicated with a filled arrow.

Figure 5

Identification of the 65 kDa PLEBP as U2AF. (A) A silver stained SDS–PAGE is shown for the final step in the purification of 65 kDa PLEBP. Input (lane 2) is the peak fractions recovered from the Phenyl-Superose column. The input sample (lane 2) and each of the recovered fractions was assayed for binding activity in (B) by UV cross-linking to the 5′-³²P-labeled PLE B RNA. In (C) and (D) each of the column fractions was assayed by western blot using polyclonal antibodies to U2AF65 and U2AF35, respectively. In (E) the column fractions were analyzed by western blot using the RS domain-specific monoclonal antibody 16H3.

Figure 6

Impact of U2AF65 and PAP-interacting domain deletions on PLE regulation of poly(A) tail length. (A) Plasmids expressing wild-type U2AF65 or U2AF65 deleted for amino acids 17–27 (Δ17–27) or 17–47 (Δ17–47) were transfected into CHO cells and expression was analyzed by western blot using a polyclonal antibody to human U2AF65. In lane 1 cells were transfected with pcDNA3 alone. (B) HeLa S3 (Tet-Off) cells were transfected in tetracycline-containing medium with the indicated U2AF65 constructs plus tetracycline-regulated plasmids bearing human β-globin reporter genes that lack (SPA) or contain a PLE (PLE). Poly(A) tail length was analyzed by RT–PCR on RNA isolated 30 h after transfection and 6 h after removing tetracycline from the medium. The center lane (lane 5) contains a marker of Hinf φX174 DNA fragments. (C) The graphing function of the ImageQuant™ program was used to determine the distribution of radioactivity in each of the lanes in (B). Note that the scales for control and PLE-containing mRNAs are different.

Figure 7

Impact of deleting the RNA-binding domains from U2AF65. The three RRM motifs were deleted from U2AF65 to generate U2AF65ΔRRM. This was cloned into pcDNA3 with an N-terminal myc tag and cells were transfected as described in Figure 6. (A) Western blot with a monoclonal antibody to the myc epitope. Lanes 1 and 3 correspond to cells transfected with pcDNA3 vector only (–) and lanes 2 and 4 to cells transfected with plasmid expressing U2AF65ΔRRM. (B) Poly(A) tail length was analyzed by RT–PCR on RNA isolated 6 h after induction as described in Figure 6C. Lanes 2 and 3 show the poly(A) tail length for RNA expressed from the control plasmid without (–, lane 2) or with (+, lane 3) co-transfected U2AF65ΔRRM. Lanes 4 and 5 show the same analysis performed on RNA expressed from the PLE-containing plasmid (PLE).

Figure 8

Impact of the C14G mutation on poly(A) tail length. (A) The sequences of the wild-type PLE, the inactive MutG element and the C14G mutation are shown aligned, with the changes from the wild-type element identified in bold. (B) Poly(A) tail length was determined by RT–PCR as in Figure 6 and equal amounts of radiolabeled products were applied to the gel. Lane 1 (M) contains a marker of Hinf φX174 DNA fragments. (C) The graphing function of the ImageQuant™ program was used to determine the distribution of radioactivity in each of the lanes for PLE-containing mRNA.

Figure 9

Impact of overexpressing U2AF65 on polyadenylation of mRNA with the C14G mutation. HeLa S3 (Tet-Off™) cells were transfected as in Figure 6 with tetracycline-regulated plasmids bearing the PLE or C14G element in the last exon of the β-globin reporter gene plus empty vector (pcDNA) and CMV-driven plasmids expressing full-length U2AF65 or the Δ17–47 mutant form of U2AF lacking the PAP-interacting domain. (A) Poly(A) tail length was determined by RT–PCR as in Figure 6 and equal amounts of radiolabeled products were applied to the gel. Lane 1 (M) contains a marker of Hinf φX174 DNA fragments. (B) The graphing function of the ImageQuant™ program was used to determine the distribution of radioactivity in each of the lanes for PLE-containing mRNA. The dashed line corresponds to cells transfected with the Δ17–47 form of U2AF65.