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Nucleic Acids Research, 2003, Vol. 31, No. 21 6264-6271
© 2003 Oxford University Press

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

Haidong Gu and Daniel R. Schoenberg*

Department of Molecular and Cellular Biochemistry and the Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA

*To whom correspondence should be addressed at Department of Molecular and Cellular Biochemistry, The Ohio State University, 1645 Neil Avenue, Columbus, OH 43210-1218, USA. Tel: +1 614 688 3012; Fax: +1 614 292 4118; Email: schoenberg.3{at}osu.edu
Present address:
Haidong Gu, Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA

Received June 30, 2003; Revised August 29, 2003; Accepted September 9, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The 3' poly(A) tail on most vertebrate mRNAs plays a key role in pre-mRNA processing, export, translation and turnover. Polyadenylation is intimately linked to transcription termination (1) and mutations that inactivate 3' processing inhibit release of the processed mRNA from the site of transcription (2). Pre-mRNA 3' processing functions to define the terminal exon (3) and upstream splicing events are impacted by selection of alternative 3' processing sites (4). Polyadenylation is required for nuclear export (5) and cytoplasmic shortening of the poly(A) tail precedes the degradation of many unstable mRNAs (6,7). Finally, poly(A) acts as a length-dependent enhancer of translation initiation, where it functions both to accentuate cap-dependent translation initiation (8–10) and to help recruit the 60S ribosomal subunit to the pre-initiation complex (11).

For most vertebrate mRNAs poly(A) addition proceeds through two steps. After cleavage of the nascent transcript poly(A) polymerase (PAP) adds 10 or more resides in a slow, distributive manner. At this point the oligoadenylate tail is bound by poly(A)-binding protein II (PAB II or PABPN1) and poly(A) addition shifts to a processive reaction culminating in the addition of 200–250 residues (12,13). Shortening of this poly(A) tail during subsequent steps in mRNA metabolism results in the heterogeneous 50–200 nt poly(A) observed on numerous mRNAs.

Unlike the mRNAs described above, the Xenopus serum albumin mRNA has a discrete 17 nt poly(A) tail (14,15). This short poly(A) tail is present on both intron-containing nuclear pre-mRNA and the fully processed mRNA, suggesting that poly(A) length control occurred during the process of poly(A) addition rather than as a result of shortening of a longer poly(A) tail. Subsequent work identified two related sequence elements upstream of AAUAAA in the terminal exon of the albumin gene that can act independently to restrict the length of the poly(A) tail on reporter mRNAs to <20 nt (16). A number of other poly(A)-limiting element (PLE)-containing mRNAs were previously described (17) and the broad scope of the short poly(A) phenotype was recently confirmed by Choi and Hagedorn (18), using microarray analysis to identify mRNAs showing differential recovery by binding to oligo(dT) versus a modified eIF4E.

To function in regulating poly(A) length the PLE must be in the terminal exon. Moving the element into either an upstream intron or exon resulted in mRNAs with long, heterogeneous poly(A) tails (19). Poly(A) length control is independent of the splicing of upstream introns and deleting either of the upstream introns of a ß-globin reporter mRNA or replacing intron II with a 21 nt polypyrimidine tract had no effect on the length of the poly(A) tail of PLE-containing mRNAs. Most of our work has focused on PLE B, since that element is conserved in the 3' end of numerous mRNAs that have <20 nt poly(A) (17). The sequence of PLE B is 5'-AAAGUUC CUUCAGCUGAAAAGAG, of which the eight purines at the 3' terminus appear dispensable (19). However, changing every other pyrimidine to a purine in the UUCCUU sequence inactivated poly(A) length control, indicating an important role for this pyrimidine-rich tract. UV cross-linking identified a 65 kDa PLE-binding protein (PLEBP) that binds to an RNA bearing the native PLE but not an RNA of the same length bearing the above mutations (19).

We describe here the identification of the 65 kDa PLEBP as the large subunit of U2 snRNP auxiliary factor (U2AF). U2AF has previously been shown to define 3' splice sites (20–24) and to link 3' processing to terminal intron splicing through its interaction with PAP (25,26). Overexpressing U2AF65 or a mutant lacking the N-terminal PAP-interacting domain disrupted PLE regulation of poly(A) tail length. However, no effect was observed if the C-terminal RNA recognition motifs (RRMs) were deleted. A mutation aimed at improving U2AF binding shifted poly(A) tail length to ~50 nt and overexpressing U2AF65 returned poly(A) tail length to <20 nt. These data indicate that U2AF modulates the PLE regulation of poly(A) tail length, perhaps by recruiting another protein(s) to the PLE.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids
All of the plasmids described here are based on the previously described human ß-globin gene expression vectors CMV-glo-SPA, CMV-glo-PLE-SPA and CMV-glo-MutG-SPA (16,19). The C14G PLE mutation was prepared by site-directed mutagenesis (GeneEditorTM; Promega) using the primer 5'-AAAAGTTCCTTCAGGTGAAAAGAGCTCTAGAG. pTet OßAcLuc3 was provided by Jose Garcia-Sanz (Universidad Autãnoma, Madrid, Spain). To generate tetracycline-inducible globin constructs pTet-glo-SPA and pTet-glo-PLE-SPA, CMV-glo-SPA and CMV-glo-PLE-SPA were digested with HindIII, end-filled with Klenow fragment DNA polymerase and then digested with NcoI. The globin-SPA or globin-PLE-SPA fragments were gel purified and cloned into the above plasmid in which the luciferase insert was removed by digesting with BamHI followed by end-filling with Klenow fragment DNA polymerase plus NcoI.

The plasmid pET9c-U2AF65 for bacterial expression of wild-type U2AF65 was generously provided by Adrian Krainer. Using it as template, PCR was performed with primers 5'-GCAGGTACCAGCATGGCGGATTTTGATGAGTTTGAG and 5'-GCAGGATCCCTACCAGAAGTCCCG and Pfu DNA polymerase. The PCR product was digested with KpnI and BamHI and then cloned between the KpnI and BamHI sites of pcDNA3. Using this pcDNA3-U2AF65 as a template, the PAP-interacting domain was removed by GeneEditorTM site-directed mutagenesis system, with primer 5'-TCAACGAGAATAAACAAAGCCACAGCCGCTCTCG or 5'-TCAACGAGAATAAACAAAACCGGGACCAGCGGAG, to generate pcDNA3/{Delta}17–27 and pcDNA3/{Delta}17–47. The N-terminal portion of U2AF65 spanning amino acids 1–153 bearing the PAP- and U2AF35-interacting domains was PCR amplified with primers 5'-GCAGGCCATGGAGGCCGCGGATTTTGATGAGTT and 5'-GCAGCGGCCGCCTACACGTAGAGGCGCCGGG. This was digested with SfiI and NotI and inserted into a modified pcDNA3 plasmid carrying an N-terminal myc eptitope tag.

Cell transfection, RNA isolation and poly(A) length assay
LM(tk–) cells were obtained from the American Type Culture Collection and maintained in Dulbecco’s minimal essential medium (DMEM) with 5% fetal calf serum (FBS) and 2 mM glutamine. One day before transfection, 8 x 105 cells were seeded into 60 mm dishes. Cells were transfected with 5 µg of plasmid DNA plus 30 µl of SuperfectTM (Qiagen) following the manufacturer’s recommended protocol. RNA was harvested 24 or 48 h after transfecton using TrizolTM reagent (Invitrogen). RNA recovered in this manner was washed with 75% ethanol, air dried, resuspended in water and stored at –80°C. Poly(A) tail length was assayed by RT–PCR as described previously (16) using the unlabeled primer 5'-GGGGATCCGCGGTTTTTTTTTT for reverse transcription and PCR and the 5'-32P-labeled primer 5'-GGCAACGTGCTGGTCTGTGT for globin exon 3. The recovered products were normalized to equal counts and separated by electrophoresis on 6% polyacrylamide/urea gels. Poly(A) tail length distribution was determined by phosphorimager analysis and the graphical distribution of poly(A) lengths relative to mobility markers was determined using ImageQuant 5.2TM software (Molecular Dynamics).

Tet-Off HeLa S3 cells and CHO cells were purchased from Clontech and maintained in DMEM with 10% FBS, 4 mM glutamine and 100 µg/ml G418 (Invitrogen). One day before transfection, 3 x 106 cells were seeded into 100 mm dishes with 100 µg/ml G418 and 1 µg/ml tetracycline added to the medium. Cells were transfected with 20 µg of U2AF65 constructs (wild-type, {Delta}17–27, {Delta}17–47, {Delta}RRM or pcDNA3 vector) and 4 µg of Tet-globin constructs (pTet-glo-SPA or pTet-glo-PLE-SPA), using TransIT®-HeLaMONSTERTM transfection reagent (Mirus Corp.). The cells were incubated with 1 µg/ml tetracycline for 24 h before the withdrawal of tetracycline. Protein and RNA were harvested after 7 h of induction and poly(A) analysis and western blotting were performed as described below.

Purification of U2AF from HeLa nuclear extract
Packed HeLa S3 cells were obtained from the National Cell Culture Center. HeLa nuclear extract was prepared as described by Wahle and Keller (27). PLE-binding activity was assayed throughout the separation by UV cross-linking, SDS–PAGE and autoradiography (see below) and the degree of purification was determined by silver stained SDS–PAGE gels of individual column fractions. The Core buffer used throughout the purification contained 20 mM HEPES–KOH, pH 7.9, 0.5 mM EDTA, 10% glycerol, 0.02% NP-40, 0.5 mM dithiothreitol and 0.5 mM phenylmethylsulfonyl fluoride. (NH4)2SO4 concentrations were adjusted to this Core buffer accordingly. Aliquots of 30 ml of HeLa cell total nuclear extract was dialysed overnight against Core buffer containing 40 mM (NH4)2SO4. This was loaded onto a 1.5 x 30 cm DEAE fast flow Sepharose ion exchange column followed by washing with 2 bed volumes of loading buffer and a 45 ml gradient of 40–350 mM (NH4)2SO4. Subsequent purification steps were performed using a Pharmacia FPLC. PLE-binding activity eluted at 70–110 mM (NH4)2SO4 from the DEAE column. The peak fractions were pooled, dialysed against Core buffer containing 20 mM (NH4)2SO4 and applied to a Mono S ion exchange column (HR 5/5; Pharmacia). The column was washed with 7–10 bed volumes of the same buffer and then eluted with a gradient of 20–300 mM (NH4)2SO4 in Core buffer in 20 bed volumes. The peak activity from the Mono S column was pooled, dialyzed to 200 mM (NH4)2SO4 in Core buffer and applied to a heparin–Sepharose column (HiTrapTM, Pharmacia). The column was washed with 7–10 bed volumes of 200 mM (NH4)2SO4 buffer and then eluted with a gradient of 20 bed volumes of 200 mM–1 M (NH4)2SO4 in Core buffer. Specific PLE-binding activity was pooled, dialyzed against 450 mM (NH4)2SO4 in Core buffer and applied to a Phenyl-Superose column (HR 5/5; Pharmacia). The column was washed with 7–10 bed volumes of Core buffer containing 450 mM (NH4)2SO4 and eluted with a 16 bed volume gradient of 450–0 mM (NH4)2SO4. The pooled peak fractions from the Phenyl-Superose column were dialyzed against Core buffer containing 20 mM (NH4)2SO4 and applied to a Mono Q ion exchange column (HR 5/5; Pharmacia). The column was then washed with 7–10 bed volumes of the same buffer and eluted with a shallow gradient of 20 bed volumes of Core buffer containing 20–200 mM (NH4)2SO4. Starting with 400 mg of HeLa nuclear extract we recovered 1 µg of purified U2AF from the final column separation. PLEBP was identified as U2AF65 by MALDI analysis and Q-TOF sequencing of tryptic peptides at the HHMI Biopolymer/W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University.

RNase footprinting
An aliquot of 100 fmol of 5'-32P-labeled PLE B RNA was incubated with or without 100 µg of HeLa nuclear extract on ice for 30 min, followed by addition of 100 ng of RNase A or T1 and incubation at 37°C for 15 or 30 s, respectively. The reaction was terminated by extraction with phenol:HCCl3:isoamyl alcohol and the aqueous phase was mixed with an equal volume of gel loading solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue), heated for 5 min at 90°C and electrophoresed on a 12% polyacrylamide/6 M urea gel. The positions of the protected fragments were determined relative to an alkaline hydrolysate of the end-labeled RNA.

RNA EMSA and UV cross-linking assays
All RNAs were gel purified prior to use. For RNA EMSA of nuclear extract 100 fmol of 5'-end-labeled 23 nt PLE RNA was mixed with 10 µg of HeLa nuclear extract in buffer containing 2 mM Tris–HCl, pH 7.6, 0.2 mM Mg(OAc)2, 0.2 mM dithiothreitol, 14 mM KCl, 2% glycerol, 0.2 mM EDTA, 8 µM EGTA and 500 ng/µl heparin. The RNA–protein mixture was incubated on ice for 30 min, followed by addition of the indicated fold excess of unlabeled PLE RNA and another 30 min on ice. The mixtures were irradiated at 254 nm for 2 min in a UV Stratalinker® 1800 (Stratagene), then electrophoresed at 170 V, 4°C for 2–3 h on a 6% native polyacrylamide gel (acrylamide:bisacrylamide 80:1). RNA–protein complexes were visualized by autoradiography and the three bands from the lane without added competitor were excised and eluted from the dried gel. These were digested with 10 µg RNase A for 30 min at 37°C. For controls 100 fmol of 5'-end-labeled RNA was mixed with 10 µg HeLa nuclear extract was mixed as above with 5'-[32P]PLE RNA or a uniformly labeled transcript for the 5' 160 nt of albumin mRNA. These were UV cross-linked and digested with RNase A as above. RNase A digestions were terminated by addition of SDS sample buffer and heating to 100°C, followed by electrophoresis by 10% SDS–PAGE and visualized by autoradiography or by phosphorimager analysis. For the homopolymer competition experiments in Figure 3, the indicated amounts of competitor RNA were added after the first 30 min of incubation and cross-linking was performed after an additional 30 min on ice.



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  		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.

 
Western blotting and antibodies
Western blotting was performed using PVDF membrane (Millipore) under neutral conditions (25 mM Tris, 192 mM glycine, 20% methanol) at 300 mA for 3 h. Blots were blocked with 5% dry milk in 1x TBS-Tween (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5% Tween-20) for 1 h, then probed with primary antibody overnight at room temperature in 1x TBS-Tween. Secondary horseradish peroxidase-coupled anti-rabbit IgG, goat antibody to ribosomal protein S6 and horseradish peroxidase-coupled rabbit anti-goat IgG were obtained from Santa Cruz Biotechnology. Rabbit anti-U2AF35 was obtained from Brent Graveley (University of Connecticut), anti-U2AF65 was obtained from Tom Maniatis (Harvard University) and 16H3 was purchased from Zymed Laboratories.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of nuclear protein(s) binding to the PLE
Previous work showed that a 65 kDa protein could be UV cross-linked to a 23 nt RNA bearing a functional PLE, but not to RNA for an element (MutG) that was inactivated by inserting three purines at every other position within the 5' pyrimidine-rich portion of the element (19). The experiment in Figure 1 used the same 23 nt RNA to examine the footprint of protein binding to the PLE. In this experiment 5'-32P-labeled PLE B RNA was incubated with and without nuclear extract, followed by digestion with RNase A or T1. Protection was observed only over the pyrimidine-rich 5' portion of the PLE (lane 4).



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  		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).

 
We next used RNA EMSA to examine the PLE binding specificity of proteins present in HeLa nuclear extract. In this experiment nuclear extract was first incubated with 5'-[32P]PLE B RNA for 30 min, followed by addition of the indicated amounts of unlabeled PLE B RNA (Fig. 2A). Only band 2 showed competition consistent with specific binding. To identify the proteins present in each of these complexes the mixture was UV irradiated, separated by RNA EMSA and visualized by autoradiography. Protein–RNA complexes extracted from each band were then digested with RNase A and electrophoresed by SDS–PAGE (Fig. 2B). Controls included nuclear extract cross-linked to [32P]PLE B RNA (lane 1) or to a transcript from the 5' end of albumin mRNA (lane 5). The 65 kDa PLEBP was present in the complex that demonstrated the greatest degree of competable binding by EMSA in Figure 2A.



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  		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.

 
The binding of the 65 kDa protein to PLE B RNA was further characterized by homopolymer competition. In the experiment in Figure 3 nuclear extract was incubated with 5'-[32P]PLE B RNA plus a 10- or 100-fold molar excess of the indicated unlabeled homopolymers. RNA–protein complexes were analyzed by UV cross-linking, SDS–PAGE and autoradiography. Binding of the 65 kDa protein was competed most efficiently by poly(U), followed by poly(rC) and PLE B RNA. No competition was observed with poly(rA) or poly(rG).

Purification of the 65 kDa PLEBP and its identification as U2AF65
PLEBP was purified from HeLa nuclear extract by conventional chromatography using the scheme diagrammed in Figure 4A. Each fraction was assayed for PLE binding by UV cross-linking to [32P]PLE B RNA or to a 32P-labeled 23 nt oligo representing the inactive MutG element (19). This is shown for each of the peak fractions in Figure 4B. Two proteins of similar size co-fractionated up to the final Mono Q column, with the PLE-binding activity eluting in fractions 9–11 (Fig. 5A and B). MALDI TOF mass spectrometry and direct sequencing of tryptic peptides identified the 65 kDa PLEBP as the 65 kDa subunit of U2 small nuclear ribonucleoprotein auxiliary splicing factor (U2AF65). U2AF consists of a heterodimer of 65 and 35 kDa subunits, and careful examination of the stained gel in Figure 5A identified a weak negatively staining band at 35 kDa. The presence of both subunits in the PLE-binding fraction was confirmed first by western blotting using polyclonal antibodies to U2AF65 (Fig. 5C) and U2AF35 (Fig. 5D) and by western blotting with the RS domain-specific monoclonal antibody 16H3 (Fig. 5E). The latter binds only to the proteins identified above. In RNA EMSA experiments both anti-U2AF65 and 16H3 effectively supershifted complexes formed between purified U2AF and PLE B RNA (data not shown) and there was no evidence for the presence of the newly identified 26 kDa U2AF subunit (28) (data not shown).



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  		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.

 


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  		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.

 
Overexpression of U2AF65 disrupts PLE regulation of poly(A) tail length
U2AF65 and 35 are tightly bound together through a reciprocal ‘tongue and groove’ arrangement (29) and neither subunit is found free of the other. This raised the possibility that a dominant negative effect might be produced by overexpressing U2AF65 in the context of the PLE-containing reporter mRNA. The experiment in Figure 6 used either wild-type U2AF65 or U2AF65 deleted in the regions shown by Vagner et al. (25) to bind to PAP. Because these constructs lacked an epitope tag and the U2AF65 antibody was directed against the human protein, proper protein expression from the full-length and deletion constructs was demonstrated first in transiently transfected CHO cells (Fig. 6A). Endogenous U2AF65 showed minimal cross-reactivity (lane 1) and wild-type U2AF65 and the deletion constructs migrated at the expected sizes (lanes 2–4). The reporter plasmids bearing the target globin gene with (PLE B) and without a PLE (control) placed downstream of tetracycline operator elements were co-transfected with the indicated U2AF65 constructs into HeLa S3 (Tet-OffTM) cells in the presence of tetracycline to maintain the target gene in a repressed state. The goal was to express the various U2AF65 proteins prior to inducing expression of the target mRNAs. Tetracycline was removed from the medium after 24 h and poly(A) tail length was determined on RNA recovered 7 h later (Fig. 6B).



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  		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 ({Delta}17–27) or 17–47 ({Delta}17–47) 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 ß-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 {phi}X174 DNA fragments. (C) The graphing function of the ImageQuantTM 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.

 
Transfection of cells with plasmids expressing U2AF65 or U2AF65 lacking amino acids 17–27 ({Delta}17–27) or 17–47 ({Delta}17–47) had no effect on the production of heterogeneous poly(A) tails on the control reporter mRNA. This is seen both in the smear of polyadenylated species observed in each of the transfectants in Figure 6B and the distribution of radioactivity on the gel shown graphically in Figure 6C. In contrast, U2AF65, and to a lesser extent the {Delta}17–27 and {Delta}17–47 forms, had a dominant negative effect on polyadenylation of PLE-containing mRNA. Overexpressing these proteins shifted the trailing edge of the peak of polyadenylated species from baseline (seen in cells transfected with vector alone) to a population of more slowly migrating RNAs.

To determine whether the RNA-binding domain was required to inhibit PLE regulation of poly(A) tail length the experiment in Figure 6 was repeated using a construct that expresses the N-terminal 153 amino acids of U2AF65 with a myc epitope tag (U2AF65{Delta}RRM). The expression of U265{Delta}RRM is shown in Figure 7A, and the impact of its expression on poly(A) tail length is shown in Figure 7B. U2AF65 lacking the RNA-binding domains had no effect on the length of the poly(A) tail on either the control mRNA lacking a PLE or PLE-containing mRNA. Therefore, the effect observed in Figure 6 resulted from U2AF65 binding to PLE-containing mRNA.



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  		Figure 7. Impact of deleting the RNA-binding domains from U2AF65. The three RRM motifs were deleted from U2AF65 to generate U2AF65{Delta}RRM. 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 U2AF65{Delta}RRM. (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 U2AF65{Delta}RRM. Lanes 4 and 5 show the same analysis performed on RNA expressed from the PLE-containing plasmid (PLE).

 
U2AF65 restores the function of an inactivated PLE
Results in Figure 1 showed that protein(s) present in nuclear extract protected the pyrimidine-rich 5' half of the PLE from RNase A, but did not protect the purine-rich 3' portion from RNase T1 digestion. The sequence of PLE B shown in Figure 8A is similar to an intronic U2AF-binding site in that it has a series of pyrimidines followed by an AG dinucleotide. The next base (at position 14 in the PLE) is a C, and others have shown that this arrangement is less favorable for U2AF binding than a G after the AG dinucleotide (24). The sequence of the C14G mutation aligned with the wild-type PLE B and inactive MutG is shown in Figure 8A, and its impact on polyadenylation is shown in Figure 8B. In contrast to MutG, mRNA carrying the C14G mutation had poly(A) ranging from 20 to ~50 nt. A graphical representation of the radioactivity distribution in each lane in Figure 8C confirmed that this single base change disrupted PLE regulation of poly(A) tail length and produced a new pattern of polyadenylated products.



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  		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 {phi}X174 DNA fragments. (C) The graphing function of the ImageQuantTM program was used to determine the distribution of radioactivity in each of the lanes for PLE-containing mRNA.

 
The approach employed in Figure 6 was used to examine the impact of U2AF65 on the polyadenylation of mRNA bearing the C14G PLE. As in Figure 6A, U2AF65 had a dominant negative effect on PLE regulation of poly(A) tail length, shifting the product from a discrete peak to a broader distribution of polyadenylated mRNAs. Again, this effect was reduced if the PAP-interacting domain was removed ({Delta}17–47). The opposite effect was observed for mRNA bearing the C14G PLE mutation. Here, both U2AF65 and the {Delta}17–47 form of the protein shifted the 25–50 nt poly(A) tail to a discrete product characteristic of the PLE. The magnitude of this effect is seen more clearly in the graphical distribution of radioactivity shown in Figure 9B. These data indicate that U2AF can restore poly(A) length control to an altered PLE.



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  		Figure 9. Impact of overexpressing U2AF65 on polyadenylation of mRNA with the C14G mutation. HeLa S3 (Tet-OffTM) cells were transfected as in Figure 6 with tetracycline-regulated plasmids bearing the PLE or C14G element in the last exon of the ß-globin reporter gene plus empty vector (pcDNA) and CMV-driven plasmids expressing full-length U2AF65 or the {Delta}17–47 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 {phi}X174 DNA fragments. (B) The graphing function of the ImageQuantTM 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 {Delta}17–47 form of U2AF65.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The PLE contains a pyrimidine-rich stretch followed by an AG dinucleotide, a sequence motif that is similar to intronic U2AF-binding sites (21). Since U2AF65 interacts with PAP to modulate the interaction between the terminal intron and 3' processing (25,30), identification of U2AF as a PLE-binding protein provided a plausible model for PLE regulation of poly(A) tail length, where persistent binding of U2AF to the PLE in the terminal exon might inhibit the second step in poly(A) addition. However, our data do not support this model and instead show that U2AF modulates the function of the PLE.

The dominant negative effect following overexpression of U2AF65 monomer suggests that U2AF competes with another protein for binding to the PLE. Support for this comes from two additional observations. First, overexpression of U2AF65 lacking the C-terminal RRMs had no effect on PLE regulation of poly(A) tail length (Fig. 7), indicating that the effect of U2AF65 is dependent on its ability to bind to RNA. Second, reducing U2AF65 to 10% of wild-type by RNAi caused a subtle sharpening of the peak of polyadenylated PLE-containing mRNA (data not shown), consistent with the removal of a competitor for binding to the PLE. Interestingly, reducing U2AF65 to this extent had no effect on splicing of the ß-globin reporter mRNA.

Mutating the C at position 14 to a G, a change shown to improve U2AF binding in the context of the 3' splice site (22), shifted poly(A) tail length from <20 nt to a distribution of 25–50 residues (Figs 7 and 9). However, this was overcome upon expression of monomeric U2AF65 or a mutant U2AF65 lacking the PAP-interacting domain. These results suggest that the C14G mutation shifted the binding equilibrium in favor of U2AF versus a poly(A) regulatory protein and the overexpressed U2AF monomer interfered with this in a manner that restored poly(A) length control. U2AF plays a transitory role in pre-mRNA splicing, where it acts to recruit U2 snRNP to the 3' splice site. We hypothesize that U2AF may have a similar transient role in PLE regulation of poly(A) tail length, where perhaps it functions to recruit another protein(s) to the PLE. Potential candidates are proteins known to interact with U2AF, such as SR proteins or SF3b.


   ACKNOWLEDGEMENTS
 
We wish to thank Jaydip Das Gupta for his assistance with the purification of U2AF, Adrian Krainer for the U2AF65 expression plasmid, Tom Maniatis and Brent Graveley for antibodies to U2AF35 and U2AF65, Jose Garcia-Sanz for the tetracycline-regulated plasmid, the National Cell Culture Center for providing HeLa cells and the HHMI Biopolymer/W.M. Keck Resource Laboratory at Yale University for assistance with mass spectrometry. We also wish to thank Brent Graveley for his helpful discussions. This work was supported by Public Health Service grant R01 GM55407 from the National Institute of General Medical Sciences to D.R.S. Support for core facilities was provided by grant P30 CA16058 from the National Cancer Institute to The Ohio State University Comprehensive Cancer.


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 DISCUSSION
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