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J. Biol. Chem., Vol. 281, Issue 5, 2460-2469, February 3, 2006

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Arginine/Serine-rich Protein Interaction Domain-dependent Modulation of a Tau Exon 10 Splicing Enhancer

ALTERED INTERACTIONS AND MECHANISMS FOR FUNCTIONALLY ANTAGONISTIC FTDP-17 MUTATIONS 280K AND N279K^*

Ian D'Souza¹ and Gerard D. Schellenberg^¶||

From the Departments of Medicine (Division of Gerontology and Geriatric Medicine), ^¶Pharmacology and ^||Neurology, University of Washington, Seattle, Washington 98195 and the Geriatric Research Education and Clinical Center, Veterans Affairs Puget Sound Health Care System, Seattle Division, Seattle, Washington 98108

Received for publication, May 27, 2005 , and in revised form, November 2, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Tau exon 10 splicing is altered by autosomal dominant mutations that cause frontotemporal dementia with parkinsonism chromosome 17-type and by unknown mechanisms in other related neurodegenerative disorders. Identifying cis- and trans-regulators of tau exon 10 splicing is therefore crucial for understanding disease mechanisms. We previously identified several splicing enhancers and silencers within exon 10 and intron 10. Here, we show that splicing factors SF2/ASF, Tra2, and a 50-kDa nuclear protein bind in vitro to the polypurine enhancer at the 5' end of exon 10. Disease splicing mutations N279K and 280K disrupt the enhancer and alter associations with these factors. N279K targets robustly bind Tra2 compared with the normal enhancer, which may explain why N279K enhances exon 10 splicing in vivo. In contrast, factor associations with 280K targets are nearly undetectable, explaining why 280K almost abolishes exon 10 splicing in vivo. Small interfering RNA-mediated suppression of endogenous SF2/ASF and Tra2 significantly reduces exon 10 splicing. Exogenous SF2/ASF dramatically enhances normal exon 10 splicing and efficiently rescues the 280K splicing defect. Domain deletion analyses show that the C-terminal RS domains of SF2/ASF and Tra2 are required for normal exon 10 splicing in vivo. In contrast to Tra2, the SF2/ASF RS domain remains essential in the presence of a strengthened enhancer or when either weak splice site is strengthened. The data suggest that SF2/ASF has both essential and regulatory roles, whereas Tra2 has a supporting role in exon 10 splicing.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In the central nervous system, the microtubule-associated protein tau is highly expressed in neuronal axons and at lower levels in glial cells. Tau functions in microtubule assembly, influences microtubule stability and is important for neurogenesis, axonal maintenance, and axonal transport (1, 2). In the human tau gene (MAPT), exons 9–12 each encode a 31–32-amino acid imperfect repeat that comprises the microtubule-binding domain of tau. In adult human brain, exons 2, 3, and 10 are alternatively spliced to produce six different tau isoforms (3–5). Exon 10 (E10)² inclusion generates isoforms with four microtubule repeats called four-repeat (4R) tau. When E10 is skipped, the result is 3R tau isoforms. The 4R/3R ratio in normal adult brain is 1. In fetal brain, only the shortest 3R tau isoform is expressed because of constitutive exclusion of exons 2, 3, and 10 (6, 7).

Pre-mRNA splicing regulation is complex requiring multiple interactions between small ribonucleoprotein particles and non-small ribonucleoprotein particle splicing factors with conserved and nonconserved splicing elements in the unprocessed RNA transcript (8). Conserved elements are the 3' and 5' splice site signals that demarcate the exonintron boundary. Nonconserved sequences that promote splicing are called exon splicing enhancers (ESE) and intron splicing enhancers (ISE) (9). Serine/arginine-rich (SR) proteins usually bind to enhancer elements and are essential at multiple steps in the splicing pathway (10). Most alternatively spliced exons contain a weak splice site, whose recognition requires SR factor associations with ESEs and ISEs (11). Alternative splicing is also influenced by splicing inhibitory sequences called exon splicing silencers and intron splicing silencers. An additional function of some enhancers is to counteract the inhibitory effects of neighboring silencer elements (12–17).

For MAPT, cis-regulation of E10 alternative splicing has been extensively studied, where multiple enhancers and silencers regulate use of the weak E10 3' and 5' splice sites (14, 18). E10 sequences include three nonredundant, weak ESEs in the first half of the exon that include an SC35-like ESE, a polypurine enhancer (PPE), and an A/C-rich enhancer separated by a central exon splicing silencer from additional ESE sequences at the 3' end of E10. Intronic sequences immediately downstream of the E10 5' splice site include an intron splicing silencer and an adjacent intron splicing modulator, which counteracts the intron splicing silencer.

Mutations in MAPT cause frontotemporal dementia with parkinsonism chromosome 17-type (FTDP-17), an autosomal dominant neurodegenerative disease (19–22). FTDP-17 belongs a group of disorders called tauopathies, in which hyperphosphorylated tau forms pathological neuronal aggregates called neurofibrillary tangles. Other tauopathies include Alzheimer disease, progressive supranuclear palsy, corticobasal degeneration, and Pick disease. Over 30 coding and intronic MAPT mutations are known that cause FTDP-17 (23, 24). One class includes missense mutations that alter tau protein function. The second mutation class includes missense, silent, deletion, and intronic mutations that alter E10 splicing by disrupting either the 5' splice site (14, 21, 25), PPE, A/C-rich enhancer, and exon splicing silencer sequences in E10 (18, 26–29) or the intron splicing silencer and intron splicing modulator sequences in intron 10 (I10) (14, 30). Most splicing mutations increase 4R tau isoforms and alter the 4R/3R ratio from 1 to 2–3. However, mutations 280K and E10+19, which decrease E10 inclusion in splicing assays, are expected to reduce the 4R/3R ratio to as low as 0.33. Silent and intronic mutations do not change the protein sequence of tau but affect E10 splicing and ultimately tau function. Thus, subtle changes in the ratio of normal tau isoforms cause severe neurodegeneration.

Identifying proteins that associate with cis-elements is necessary to elucidate how E10 splicing is regulated. Here we focus on identifying protein interactions with the E10 PPE, a 9-nucleotide purine-rich ESE sequence between E10 positions +16 and +24 (see Fig. 1A). The PPE sequence contains two copies of an AAG motif and one copy of a GAR (where R is a purine) motif that occur frequently in natural, purine-rich ESEs. These motifs are often high affinity binding sites for SR and SR-related proteins. FTDP-17 mutations N279K and 280K alter the normal PPE sequence by adding or removing an AAG copy, respectively (see Fig. 1A). Consequently these mutations have opposite effects on E10 splicing, where N279K increases and 280K decreases E10 inclusion. Our earlier work predicted that the normal PPE function requires SR factor interactions, which are altered by disease mutations 280K and N279K (26). Indeed, the SR-like splicing factor Tra2 is essential for E10 inclusion in splicing assays and associates in vitro with both normal and mutant PPE templates (31). Here we show specific in vitro associations of a trio of factors with the normal and strengthened (mutation N279K) PPE but not with a disrupted PPE (mutation 280K) and identify two of these proteins as the SR domain-containing factors SF2/ASF and Tra2. We corroborate their specific effects on E10 splicing by gene knock-down assays and present in vivo mechanisms for their varied effects on E10 splicing. Overexpression of SF2/ASF rescues the splicing defect in mutation 280K. Domain deletion assays in non-neuronal and neuronal cells show that the C-terminal protein-interacting domains of both SF2/ASF and Tra2 are required for E10 splicing. Our data reveal PPE-dependent and PPE-independent roles for SF2/ASF for use of the weak 3' and 5' splice sites, respectively. Thus, SF2/ASF has complex essential and regulatory roles on E10 splicing, whereas the role of Tra2 appears secondary to SF2/ASF.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Plasmid Construction and DNA Mutagenesis—Splicing vector hN has been described (18) and contains the entire 93-bp MAPT E10 sequence with 33 and 51 bp of flanking introns (see Fig. 3A). Minigene construct E10AH (see Fig. 2) contains entire MAPT exons 9, 10, and 11 as well as 324 and 559 bp of intron 9 and I10 sequences, respectively, inserted into expression vector pRcRSV (Invitrogen). Intron 9 in E10AH contains 224 bp of sequence immediately 5' of E9 and 100 bp of sequence immediately 3' of E10. I10 in E10AH contains 100 bp of sequence immediately 5' of E10 and 459 bp of sequence immediately 5' of E11. FTDP-17 mutations 280K and N279K were introduced into hN (18) and E10AH by PCR mutagenesis.

SF2/ASF, Tra2, and hnRNP G Expression Constructs—SF2/ASF cDNA was amplified by RT-PCR from HeLa RNA using primers ASF5'F (5'-AGCTGGATCCATGTCGGGAGGTGGTG-3') and ASF3'R (5'-CGATCTCGAGTTATGTACGAGAGCGAGATCTGC-3'). The PCR product was digested at BamHI and XhoI sites in the primer sequences and inserted into vector pcDNA3.1/Zeo+ (Invitrogen) to generate expression construct SF2/ASF. The Tra2 expression vector contains a cDNA sequence obtained by PCR amplification from vector Htra2-1-V5 (32) using primers Tra2b5'F (5'-AGCTGGATCCATGAGCGACAGCGGCGGCAG-3') and Tra2b3'R (5'-CGATCTCGAGTTAATAGCGACGAGGTGAGTATGATCG-3'). The BamHI/XhoI-digested product was inserted into pcDNA3.1/Zeo+. We generated domain deletion constructs by PCR mutagenesis of full-length SF2/ASF and Tra2 expression vectors (see Fig. 4A). Primers used to delete the N-terminal RRM domains from SF2/ASF (construct SF2/ASF-RRM) were SF2dRRMF (5'-AGCTGGATCCATGCGTGAAGCAGGTGATGTA-3') and SF23'R. For RS domain deletion construct SF2/ASF-RS, the primer pair SF25'F and SF2dRS (5'-GCGCTCGAGTTATGGGCCCATCAACTTTAACC-3') was used. The N-terminal RS domain in construct Tra2-RS1 (see Fig. 4A) was deleted using primers Tra2dRS1 (5'-CGCGGATCCATGCATGTTGGGAATCGGGCAAATCC-3') and Tra2b3'R. The central RRM domain in Tra2-RRM (see Fig. 4A) was deleted using primers Tra2b5'F and Tra2b3'R with expression construct pCMVhtl1 + 5' as template (kind gift from William Mattox). In Tra2-RS2 (see Fig. 4A) the C-terminal RS domain was deleted using primers Tra2b5'F and Tra2bdRS2 (5'-GCGCTCGAGTTATCCTGGTGTTGGCGTATGTGG-3'). Construct hnRNP-G-V5 is described elsewhere (32).

Cell Culture, Transfections, and RNA Isolation—Maintenance and transfection of PC12 cells is described elsewhere (14). HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen). All of the transfections were performed in triplicate with Lipfectamine (Invitrogen) in six-well plates. HeLa cells were transfected with 2 µg of plasmid DNA and 6 µl of Lipofectamine in 1 ml of OptiMEM (Invitrogen) for 5 h at 37 °C (5% CO₂), after which 1 ml of Dulbecco's modified Eagle's medium supplemented with 20% fetal bovine serum was added. For coexpression experiments in PC12 cells, 2.5 µg of total plasmid containing 0.5 µgof an E10 splicing vector and 2 µg (4-fold excess) of either pSKII control vector (Stratagene) or individual splicing factor expression constructs were used with 6 µl of Lipofectamine. Total RNA was isolated with TRIzol reagent (Invitrogen) as previously described (26).

siRNA Synthesis and Transfection—Commercially synthesized (Dharmacon Inc.) siCONTROL nontargeting siRNA number 1 and gene-specific siRNA pools were used for endogenous suppression of SF2/ASF and Tra2. siRNA transfections were optimized in 24-well plates containing cells at 70% confluency using Oligofectamine (Invitrogen) with 25, 50, 75, and 100 nmol of each siRNA in the pool (see Fig. 2A). Maximal inhibition was observed with 100 nmol of each siRNA. Protein was harvested 48–72 h post-transfection, and expression of targeted factors was tested by immunoblot analyses (described below). To test the effects of siRNA treatment on E10 splicing vectors, the cells were seeded into 12-well plates, and siRNA transfections were scaled up. After 24 h, the cells were rinsed in phosphate-buffered saline and fed 450 µl of antibiotic-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. One µg of normal or mutant splicing vector E10AH was mixed with 3 µl of Lipofectamine in 50 µlof OptiMEM and added to each well. Total RNA was isolated 48 h later for RT-PCR analyses with TRIzol reagent (Invitrogen).

Quantitation of Tau E10 Splicing by RT-PCR—Tau E10 splicing from transiently transfected splicing vectors was analyzed by RT-PCR assays as described previously (26). E10+ and E10– transcripts were amplified by RT-PCR using vector-specific primer pairs SD6/SA2 for hN and pREP/BGHPA2 for E10AH as previously reported (18, 33). Amplified products from hN are 261 bp (E10–) and 354 bp (E10+) and from E10AH are 447 bp (E10–) and 540 bp (E10+). The products were resolved on 5–7.5% acrylamide gels and quantitated using a Molecular Imager® system (Bio-Rad). For each mutant construct, the values presented are the averages of at least three different transfection experiments with the normal E10 construct transfected in parallel. Statistical comparisons were made using a two-tailed Student's t test. Criteria for significance were calculated using a Bonferroni correction for multiple comparisons.

In Vitro RNA Synthesis—A primer-based strategy was used to in vitro synthesize short, radiolabeled RNAs containing normal or mutant PPE sequences. Forward and reverse primers were designed such that on annealing they form a double-stranded T7 promoter region upstream of the RNA target to be transcribed. A universal forward primer, T7F3 (5'-CGCTCTAGATAATACGACTCACTATAGGGAGTAT-3') containing the core T7 promoter sequence (italics) was used. Nine additional nucleotides upstream of the T7 promoter sequence maximize transcription efficiency, and five additional nucleotides downstream of the promoter serve as a linker that overlaps with the RNA target. The reverse primer contains the reverse complement of the core T7 promoter (underlined) with tau sequences (italics) that include the normal or mutant PPE element (bold type). The reverse primers used were: T7PPE (5'-GCCTCCTGGATCCAGCTTCTTATTATACTCCCTATAGTGAGTCGTATTAGCG-3'), T7279K (5'-GCCTCCTGGATCCAGCTTCTTCTTATACTCCCTATAGTGAGTCGTATTAGCG-3'), and T7280K (5'-GCCTCCTGGATCCAGCTTATTATACTCCCTATAGTGAGTCGTATTAGCG-3'). To anneal universal and reverse primers, 100 ng of each in 0.1 M NaCl was heated to 94 °C for 3 min and cooled at room temperature for 10 min. For in vitro transcription, 1 µlof the annealed primer reaction was added to 19 µl of a transcription mixture containing 1x T7 polymerase buffer (Invitrogen), 20 mM dithiothreitol, 20 units of RNasin (Promega), 5 µM [³²P]ATP or [³²P]GTP (800 Ci/mmol), 0.5 mM each of the remaining three NTPs and 50 units of T7 RNA polymerase. The reactions were incubated at 31 °C for 2 h, heat-denatured at 75 °C for 2 min, quenched on ice for 2 min, and treated with 10 units of DNase I at 37 °C for 20 min to remove primers. 7 µl of formamide loading dye was added to samples, heated at 95 °C for 5 min, and resolved on 12% urea-PAGE gels. Full-length transcripts were visualized by autoradiography, excised, and eluted in 400 µl of elution mix (0.5 M ammonium acetate, 2.5 mM EDTA, 0.5% SDS) for 1 h at room temperature. The eluate was ethanol-precipitated and resuspended in 100 µl of RNase-free H₂O, and 1 µl was used for quantitation in a scintillation counter.

HeLa Nuclear Extract Preparation, UV Cross-linking, and Affinity Purification—HeLa nuclear extracts were prepared as described (34). The cell pellets were obtained from HeLa-S3 spinner cultures grown at the National Cell Culture Center, a research resource facility established by the National Center of Resources for Research, National Institutes of Health.

For UV cross-linking, radiolabeled (30–100 x 10⁵ cpm) gel-purified, full-length RNA targets were incubated with HeLa nuclear extract in 10-µl reactions under splicing-permissive conditions (0.6 mM ATP, 0.1 M creatinine phosphate, 1.5 mM MgCl₂, 50–75 µg of HeLa extract, 4 µg each of tRNA and heparin, and 2 µlof10x binding buffer containing 20 mM HEPES (pH 7.9), 1 M potassium glutamate, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 20% glycerol). The reactions were incubated at 30 °C for 30 min, exposed to UV light (0.2 J for 12 min) at a distance of 4 cm on ice to form covalent RNA-protein complexes before treating with RNase A to remove unbound RNA sequences. 11 µlof2x SDS protein loading buffer was added to the samples before boiling for 5 min. The bound proteins were resolved on 10–15% SDS-PAGE gels and visualized by autoradiography.

For RNA affinity purification, 5'-Ome-modified normal PPE and mutant (280K,279K) RNA target sequences were commercially synthesized (Dharmacon Inc.) and are identical in sequence to in vitro transcribed templates used in UV cross-linking assays. RNA-agarose affinity columns were prepared according to methods described before (35) and incubated with 200 µl of HeLa nuclear extract under splicing conditions at 30 °C for 20 min to allow binding of splicing factors. Protein-RNA-agarose complexes were collected without exposure to UV light and washed extensively in Dignam Buffer D before eluting bound factors in 2x SDS gel loading buffer. The samples were boiled for 5 min, resolved on 10–12% SDS-PAGE gels, and electroblotted for immunodetection assays.

Immunoblotting—Monoclonal antibody mAb104 (1:100) was used to detect the SR family of splicing factors that range in size from 20 to 85 kDa (36). SF2/ASF was detected by a monoclonal antibody (1:5000) (37). Tra2 was detected using a polyclonal antibody Rb3505 (1:1500) raised against a synthetic peptide containing the N-terminal 15 amino acids (MSDSGEQNYGERESR) (Invitrogen Antibody Services). Anti--tubulin monoclonal antibody E7 was obtained from the NICHD Developmental Studies Hybridoma Bank maintained by The University of Iowa (Department of Biological Sciences, Iowa City, IA) and was used at dilution 1:3000 as an internal protein control. Rabbit secondary antibodies (Jackson ImmunoResearch) used at 1:2500 dilution were anti-mouse IgM for mAb104 and anti-mouse IgG for both SF2/ASF and -tubulin. Chemiluminescent immunodetection was achieved with horseradish peroxidase conjugated to protein A (Sigma) at a dilution of 1:3000 and the ECL plus detection system (Amersham Biosciences).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The following experiments were designed to identify the trans-acting proteins that recognize the normal PPE, to determine whether this protein-RNA interaction is disrupted by mutations that eliminate E10 inclusion, and to determine whether PPE mutations that enhance E10 inclusion work by strengthening normal protein-RNA interactions or cause different proteins to interact with the extended PPE.

Altered Association of Splicing Factors with Normal and Mutant PPE Sequences—To identify proteins associations, radiolabeled RNA targets containing the normal and mutant PPE sequences (Fig. 1A) were incubated with HeLa nuclear extracts under splicing conditions and treated with UV light to crosslink RNA-protein complexes. Because purine-rich enhancers such as the PPE bind SR proteins, we first tested four different HeLa nuclear extract preparations for SR protein content by immunoblot analysis. The pan-SR monoclonal antibody mAb104 recognizes phosphorylated epitopes within the RS domain of prominent SR protein family members SRp75, SRp55, SRp40, and SRp30 (36) that were consistently observed in all four nuclear extract preparations (Fig. 1B). UV cross-linking experiments reveal three proteins with apparent molecular masses of 52, 45, and 36 kDa that individually cross-link to normal PPE and 279K RNA targets (Fig. 1C). The 45-kDa factor interacts more strongly with 279K than normal PPE templates. In contrast, almost no SR protein cross-links to the 280K target, although overexposure of the immunoblot showed very weak signals from the 52- and 36-kDa factors but no signal from the 45-kDa factor (not shown). Similar results were obtained with different HeLa extract preparations. Thus, mutation 280K may inhibit E10 splicing by abolishing productive SR protein interactions. The near absence of detectable proteins with 280K targets verifies the binding specificity and rules out nonspecific interactions with flanking linker sequences. Candidate proteins for the 45-kDa band are SR factor SRp40 and the SR-like factor Tra2. Based on molecular size, the candidates for the 36-kDa protein are SF2/ASF (SRp30a), SC35 (SRp30b), and SRp30c.

To identify these proteins, RNA affinity columns were constructed by coupling the normal and mutant RNA sequences to agarose. Columns were incubated with HeLa nuclear extract under splicing conditions as above but without exposure to UV light. Bound proteins were eluted and analyzed by immunoblotting with Tra2- and SF2/ASF-specific antibodies. We probed for Tra2 because it preferentially binds AAG repeats (38), present in two and three copies, respectively, in normal and 279K templates (Fig. 1A). Consistent with this observation and similar to the binding profiles in UV cross-linking assays, we identify the 45- and 36-kDa proteins as Tra2 and SF2/ASF, respectively. Tra2 eluted robustly from the 279K column compared with the normal PPE column (Fig. 1D) and SF2/ASF eluted at similar levels from both columns. Interestingly, only SF2/ASF was eluted at very low levels from 280K. The in vitro affinity of SF2/ASF and Tra2 for normal PPE and 279K mutant templates correlates well with their match to their respective binding consensus sequences (Fig. 1A). Missense mutation N279K strengthens the SF2/ASF binding consensus and may allow stronger SF2/ASF binding, although the observed amounts of SF2/ASF bound to 279K and normal PPE targets are similar. ESE sequences are usually degenerate to accommodate their presence within diverse coding sequences. This degeneracy allows more than one SR protein to recognize the same ESE (39, 40). Our results show that the normal PPE sequence is an overlapping low affinity site for both SF2/ASF and Tra2. Other examples have been described where SF2/ASF associates with an ESE that is also recognized by Tra2 (41). The identity of the 50-kDa factor detected in the UV cross-linking experiments is unknown and is being pursued. In addition to mutation N279K (AAT AAG), we previously showed that a different transversion (AAT AAA) at the same nucleotide position strengthened the PPE by increasing the number of consecutive purines to enhance E10 splicing (18). Because these substitutions enhanced E10 splicing to different extents, the purine context is an important determinant of PPE strength. Although the T to A change increases the number of consecutive purines, unlike in N279K, it does not add an extra AAG copy and is less efficient in E10 enhancement, possibly because of a decreased affinity for Tra2.

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Identification of nuclear factors that bind normal and mutant PPE targets. A, sequences of in vitro transcribed normal (PPE) and mutant (280K,279K) RNA targets. E10 sequences (bold type) contain the nine-nucleotide core PPE element (underlined) and are flanked by 8 nucleotides of linker sequence shown in normal letters. Deletion mutation 280K removes an AAG copy (dashed line) from the PPE. Missense mutation N279K (arrow) creates an extra AAG copy and extends the 5'-end of the PPE. To the right are shown differences in the match of normal and mutant PPE sequences to the 8-nucleotide preferred binding consensus for SF2/ASF and in the amounts of AAG or GAR (where R is a purine) repeats favored by Tra2. B, immunoblot analysis of four separate HeLa nuclear extract preparations (lanes A–D) using monoclonal antibody mAb104, which detects members of the SR family ranging from 30 to 70 kDa. C, autoradiograph of UV cross-linking assays showing 52-, 45-, and 36-kDa factors covalently bound to radiolabeled normal (PPE) and mutant (279K) targets. Associations with 280K templates are virtually undetectable. D, immunoblot analysis of HeLa nuclear factors eluted from 280K, PPE, and 279K RNA-agarose affinity columns. The Tra2 polyclonal antibody Rb3505 and the SF2/ASF-specific monoclonal antibody identify 45- and 36-kDa bands (arrows, right panel), respectively, from normal and 279K RNA columns. Only SF2/ASF is weakly detected from 280K affinity columns.

Overexpression of Candidate Splicing Factors in PC12 and HeLa Cells—The effects of candidate factors on E10 splicing were tested in vivo by overexpressing these proteins in cells cotransfected with either E10AH or another E10 splicing vector hN, in which E10 is inserted in an intron between two heterologous exons (Fig. 3A). E10 splicing in E10AH is similar in both transiently transfected HeLa (76%) and PC12 (82%) cells (Fig. 3B). In both cell types, mutation N279K causes almost constitutive E10 inclusion (90–93%), whereas mutation 280K severely inhibits E10 splicing (5–14%). As shown previously for hN, normal E10 inclusion is 45% in COS-7, PC12, and rat P1 neurons, 79% for N279K (COS-7 cells), and <5% for 280K (COS-7 cells) (14, 18). Similar results for N279K and 280K in vector hN are also seen in HeLa and PC12 cells (data not shown).

View larger version (40K): [in this window] [in a new window]   FIGURE 2. Knock-down effects of SF2/ASF and Tra2 on E10 splicing. A, immunoblots of HeLa cells treated with increasing concentrations of siR-NAs show reduction of endogenous SF2/ASF (si-SF2/ASF, left panel) and Tra2 (si-Tra2, middle panel) compared with -tubulin expression. The siRNA concentrations used per lane are 25, 50, 75, and 100 nmol for si-SF2/ASF and 50, 75, and 100 nmol for si-Tra2. si-SF2/ASF and si-Tra2 show gene specificity (right panel, lanes 3 and 4). The control siRNA (siCONTROL nontargeting siRNA number 1; Dharmacon, Inc.) lacks identity to known gene targets and controls for possible nonspecific effects of siRNA treatment. si-SF2/ASF and si-Tra2 cotreatment (50 nmol each) decreases the levels of both factors (right panel, lane 5) less efficiently than treatments with 100 nmol of individual siRNAs. Monoclonal antibodies SF2/ASF and E7 (-tubulin) and the polyclonal antibody Rb3505 (Tra2) were used for detection. B, E10AH minigene splicing construct containing E10 separated from flanking tau exons E9 and E11 by 0.35 and 0.55 kilobase pairs of total intronic sequences, respectively. C, bar graph showing E10+ levels from triplicate transfections. The error bars represent the standard deviations. A corrected significance criteria of p < 0.0125 was used, and p values comparing E10 inclusion in siRNA-treated samples to control samples within the same cell line are represented by symbols: , p < 1 x 10–3; , p < 1 x 10–4; and , p < 1 x 10–5. D, autoradiograph of E10+ and E10– products from representative RT-PCRs obtained by transient transfection of minigene splicing vector E10AH in mock or siCON-TROL-treated HeLa and PC12 cells (left panel) as well as in individual or combined (si-SF2/ASF + si-Tra2) gene-specific siRNA-treated HeLa (middle panel) and PC12 cells (right panel).

RS Domain-dependent Regulation of E10 Splicing—SR family proteins like SF2/ASF (Fig. 4A) contain modular functional domains that include one or two N-terminal RRM domains and a C-terminal RS domain (10). SR-related proteins like Tra2 contain a central RRM domain flanked on either side by an RS domain (Fig. 4A). To determine whether the regulatory function of a candidate factor is dependent on RNA binding or protein interaction, RRM or RS domain deletion constructs for SF2/ASF and Tra2 were generated (Fig. 4A). We cotransfected PC12 cells with a 4-fold excess of intact or domain deletion splicing factor constructs and individual minigene constructs E10AH, E10AH/280K, or E10AH/279K. 4-fold excess empty vector pSKII was cotransfected with each minigene construct as a control (Fig. 4B, top gel, lanes 1 and 9). As in Fig. 3C, E10 splicing from E10AH (77% E10+) is not altered in the presence of excess SF2/ASF (82%) or Tra2 (78%) (Fig. 4B). Deletion of both SF2/ASF RRM domains in SF2-RRM slightly reduces E10AH splicing (from 77% to 67%), whereas deletion of the protein-interacting domain in SF2-RS dramatically reduces E10AH splicing to 53% E10 inclusion. Analyses with Tra2 deletion mutants show that removal of the Tra2 RRM domain (Tra2-RRM, 77%) or N-terminal RS domain (Tra2-RS1, 77%) are neutral on E10AH splicing. However, E10AH splicing is significantly reduced (from 78 to 53%; TRa2-RS2) in the absence of the Tra2 C-terminal RS domain.

View larger version (34K): [in this window] [in a new window]   FIGURE 3. Overexpression effects of SF2/ASF and Tra2 on E10 splicing. A, the hN heterologous splicing construct is shown with 177 bp of a tau E10 genomic fragment inserted between flanking globin-tat chimeric exons. B, autoradiograph showing E10+ and E10– products in HeLa and PC12 cells transiently transfected with E10AH, E10AH/280K, and E10AH/279K constructs. The bar graph on the right shows quantitation of E10+ transcripts from triplicate RT-PCR assays. Corrected significance criteria of p < 0.025 (PC12) and p < 0.016 (HeLa) were used for comparisons of mutant with normal E10 constructs in the same cell line with significance levels as indicated in Fig. 2 including +, p < 1 x 10–2. C, bar graph showing E10+ levels in vectors hN, E10AH, and E10AH/280K coexpressed with either 4-fold excess pSKII control vector or individual expression vectors SF2/ASF, Tra2, and hnRNPG or with both expression vectors SF2/ASF and Tra2 in neuronal PC12 cells. A corrected significance criteria of p < 0.025 was used. The significance levels are symbols above lines connecting the bars for different hN and E10AH/280K constructs being compared and are described in previous figure legends. D, immunoblots showing increased expression levels of transfected intact or domain deletion constructs for SF2/ASF and Tra2 compared with mock transfected HeLa cells. Antibodies used include SF2/ASF, Rb3505 (Tra2) and E7 (-tubulin) as an internal control.

View larger version (38K): [in this window] [in a new window]   FIGURE 4. Domain deletion analyses of splicing factors on E10 splicing. A, domain structure of SR factor SF2/ASF with deletions of the entire RRM or RS domains denoted by RRM and RS, respectively. In contrast, the SR-like factor Tra2 has a different domain structure and deletion of the central RRM, and individual flanking RS domains are denoted by RRM as well as RS1 and RS2, respectively. B, autoradiographs of representative E10+ and E10– RT-PCR products obtained by cotransfecting PC12 cells with minigene vectors E10AH (top gel) or E10AH/280K (bottom gel) together with 4-fold excess control vector pSKII (lane 1), intact and domain deletion SF2 (lanes 2–4) as well as Tra2 (lanes 5–8) expression vectors. Lanes 9 and 10 in the top gel correspond to RT-PCR products from E10AH/279K cotransfected with pSKII and SF2-RS. The bar graph shows E10 splicing levels determined by RT-PCR assays from triplicate transfections. The corrected significance criteria used are: p < 0.0125, for E10AH comparisons; p < 0.0166, for E10AH/280K comparisons; and p < 0.025, for E10AH/279K comparisons. C, autoradiographs of coexpression assays performed in PC12 cells are the same as in B but with mutant E10 heterologous constructs hN/279K (top gel) and hN/280K (bottom gel). Quantitation of E10 splicing levels are shown in the bar graph. The corrected significance criteria used are: p < 0.00625 for hN/279K comparisons and p < 0.025 for hN/280K comparisons. The symbols for the significance levels are as described in preceding figure legends.

View larger version (30K): [in this window] [in a new window]   FIGURE 5. Extent of RS domain dependence of the E10 5' and 3' splice site. A, incorporation of FTDP-17 mutation E10+3 (g a) at I10 position +3 into heterologous construct hN generates hN/E10+3 and results in constitutive E10 inclusion because of strengthening of the weak E10 5' splice site. In construct hN/–3T-7T, three a u changes at intron 9 positions –3, –4, and –7, immediately upstream of E10, increase the number of consecutive pyrimidines within the polypyrimidine tract and result in constitutive E10 inclusion (13). B, the bar graph shows averages of RT-PCR assays from HeLa cells co-transfected in triplicate with splicing vectors hN/E10+3orhN/–3T-7T and 4-fold excess SF2-RS or Tra2-RS2 expression constructs. A corrected significance criteria of p < 0.025 was used. Significance levels for comparisons between samples are indicated above bars by previously described symbols and include "," p <1 x 10–6. C, autoradiograph shows representative RT-PCR samples from co-transfected HeLa cells.

RS Domain-dependent Interactions with the E10 Splice Sites—We previously reported that E10 has suboptimal 3' and 5' splice sites (14, 18). A weak polypyrimidine tract contributes to the suboptimal 3' splice site, because replacing inhibitory purines with pyrimidines at positions –3, –4, and –7 upstream of E10 (–3T-7T; Fig. 5A) strengthened the polypyrimidine tract and allowed constitutive E10 splicing. However, when the PPE was disrupted by introducing mutation 280K into construct –3T-7T, constitutive splicing was either abolished or severely reduced in COS-7 and PC12 cells, respectively. In contrast, the nearly constitutive E10 splicing achieved by strengthening the weak 5' splice site using FTDP-17 mutation E10+3 (Fig. 5A) was only marginally reduced (from 97 to 87%) when the PPE was similarly disrupted. Thus, the normal PPE functions bidirectionally but is primarily required to promote use of the 3' splice site, allowing it to efficiently establish communication with its weak 5' splice site partner.

Because protein interactions with 280K templates are nearly abolished in vitro, we asked whether altered enhancer dependence of the weak E10 splice sites is mediated through protein interactions with SF2/ASF or Tra2. RS domain deletion constructs SF2/ASF-RS and Tra2-RS2 were coexpressed individually with heterologous E10 splicing constructs hN/–3T-7T or hN/E10+3 that contain the normal PPE. As in our previous reports (14, 18), E10 inclusion in the above splicing constructs is almost constitutive (Fig. 5B). Coexpression of SF2/ASF-RS with construct hN/E10+3 severely diminishes E10 splicing (from 97 to 36%). Thus, the constitutive effect of the strong 5' splice site, although shown previously to be relatively PPE-independent, strongly depends on the SF2/ASF RS domain. Coexpression of SF2/ASF-RS with construct hN/–3T-7T partially reduces E10 splicing (from 98 to 80%). Thus, strengthening the 3' splice site appears to decrease its dependence on the SF2/ASF RS domain. Because splicing of hN/–3T-7T was previously shown to require an intact PPE, interactions of endogenous SF2/ASF or of the other members in the PPE-binding complex may be strengthened in the presence of a strong 3' splice site that overrides the effect of SF2/ASF-RS. Similar assays with Tra2-RS2 had no effect on either splicing vector. These results further support both PPE-dependent and PPE-independent roles for SF2/ASF on E10 splicing regulation. Because both E10 splice sites are normally weak, the requirement for SF2/ASF is expected to be stronger.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
FTDP-17 highlights the association between splicing mutations that affect multiple cis-sequences and the pronounced variability in phenotype that is characteristic of inherited disorders. Productive interactions that positively define E10 are mediated in part by the PPE element we previously identified. Here we identify factors that associate with the PPE and extend our understanding of how the normal PPE functions and how it is disrupted by FTDP-17 mutations 280K and N279K.

The strength of an ESE is determined by its affinity for available SR factors and the activity of their RS domains (45). FTDP-17 mutations 280K and N279K highlight the physiological relevance of the AAG repeat composition, characteristic of prototypical purine-rich ESEs, on PPE strength and function. Our in vitro binding data provide an explanation for the antagonistic effects of these two mutations on E10 splicing in vivo. Furthermore, our siRNA-mediated depletion assays validate the specific in vivo requirement of both SF2/ASF and Tra2 for normal E10 splicing. These results also give the first indication that normal E10 splicing shows a stronger requirement for SF2/ASF than Tra2, because incomplete depletion of SF2/ASF strongly reduced E10 transcripts to levels achieved only by complete depletion of Tra2. The second indication is that coexpression of SF2/ASF rather than Tra2 in neuronal PC12 cells dramatically enhanced E10 splicing in heterologous constructs hN and hN/280K (2.5-fold each) as well as in minigene construct E10AH/280K (3-fold). hN, hN/280K, and E10AH/280K splice inefficiently and appear more responsive to excess SF2/ASF in contrast to vectors E10AH and E10AH/279K, which splice almost constitutively. It is interesting that SF2/ASF coexpression in vivo substantially rescued the splicing defect in hN/280K and E10AH/280K despite a disrupted PPE, whose in vitro association with SF2/ASF is barely detectable. One explanation is that enhancers increase the local concentration of specific SR factors (51), also achieved by their exogenous overexpression. It is also possible that SF2/ASF associates with other sites within E10 in addition to the PPE. The potential for multiple SF2/ASF high affinity binding sites upstream of the E10 5' splice site is predicted (not shown) by the ESEfinder program (48). Although the function of these predicted sites requires testing, in vivo analyses in PC12 cells reveal an RNA-dependent function for SF2/ASF on normal E10 splicing. Deletion of the entire SF2/ASF RRM domain marginally decreased E10AH splicing (from 77 to 67%) but had no effect on splicing of E10AH/280K and E10AH/279K. Thus, the RNA binding function of SF2/ASF is required to maintain normal E10 levels in E10AH and, when in excess, its ability to promote E10 inclusion in E10AH and hN. Clues to the PPE-independent functions of SF2/ASF lie in its ability to rescue the splicing defect in constructs E10AH/280K and hN/280K. The third indication of the important role for SF2/ASF in E10 splicing is apparent when its RS domain is deleted. SF2-RS coexpression not only reduced E10 splicing by similar levels in constructs containing a normal (E10AH) or strengthened (E10AH/279K, hN/279K) PPE but also drastically diminished splicing (by 68%) in the splicing-deficient construct E10AH/280K. Thus, SF2/ASF-mediated protein interactions are a critical function for regulated and enhanced tau E10 splicing.

In contrast to SF2/ASF, only normal E10 showed a slight but nonsignificant increase in splicing levels when coexpressed with Tra2 in vivo (E10AH; Fig. 3C). One possibility is that endogenous levels of Tra2 are functionally adequate in maintaining E10 splicing as suggested by depletion assays. Coexpressing SF2/ASF with Tra2 did not show a synergistic increase in E10 splicing (Fig. 3C), although both factors have been shown to interact in other studies and depletion of either factor alone or together reduced normal E10 splicing by similar levels in vivo (Fig. 2C). Another possibility is that Tra2 mediates its effect by interacting with a limiting factor, which remains to be identified. Also, deletion of the Tra2 RRM domain did not affect E10 splicing, suggesting either that endogenous Tra2 association with the PPE competes efficiently with Tra2-RRM to maintain normal E10 levels or that Tra2-RRM may have an RNA-independent effect and is localized by factors already bound to the PPE. Deletion of only the C-terminal RS2 domain of Tra2 specifically decreased normal E10AH splicing, indicating a requirement for protein interactions mediated specifically by this domain (Fig. 4B). The C-terminal RS deletion mutants of SF2/ASF and Tra2 may behave in a dominant-negative fashion, where they may occupy the PPE but prevent productive protein interactions. Nevertheless, the strength and type of protein interactions mediated by the RS2 domain of Tra2 are distinct from the RS domain of SF2/ASF because both show different functional specificities in regulating E10 templates with a normal or mutant PPE. Use of a weak splice site is regulated both by the availability of SR factors and the strength of RNA-protein interactions as determined by the sequence of the recognition site. Our results show that the specific requirements for SF2/ASF and Tra2 protein interactions are also distinguished when either the E10 5' or 3' splice site is strengthened. SF2/ASF shows PPE-dependent and PPE-independent functions for the 3' and 5' splice sites, respectively. SF2/ASF protein interactions are critical for use of the weak 3' splice site despite the presence of a strong 5' splice site. Strengthening the 3' splice site almost completely compensates for the lack of SF2/ASF protein interactions with the weak 5' splice site (Fig. 5). Recently, the RS domain of SF2/ASF tethered to an ESE was shown to directly bind the branchpoint sequence region in the upstream intron and promote the assembly of splicing complexes (49). An earlier report showed that the RS domain of SF2/ASF is required to recruit the U2 small ribonucleoprotein particle-associated heterodimer U2AF⁶⁵/U2AF³⁵ to introns that contain a weak 3' splice site (50). Because the requirement for the SF2/ASF RS domain is dependent on the 3' splice site strength, an essential function of SF2/ASF bound to the PPE, which is in close proximity to the 3' splice site, would be to stabilize the association of splicing machinery components with the weak polypyrimidine tract upstream of E10. On the other hand Tra2 protein interactions are required only for normal E10 splicing but may be dispensable when either splice site is strengthened. Thus, SF2/ASF and Tra2 have distinct roles in E10 splicing where SF2/ASF appears to play a primary essential role, whereas Tra2 plays a regulatory role.

The difference in normal and enhanced tau E10 inclusion may simply reflect a difference in the strength of associations at the enhancer. Because only one factor tethered to an ESE interacts with splicing machinery components (51), the preferential association of SF2/ASF versus Tra2 may present different protein-protein interaction specificities and strengths specified by their RS domains. Thus, E10 splicing may be vulnerable to subtle alterations in the levels and activities of SF2/ASF and Tra2. Our observations highlight the requirement of SF2/ASF for use of the weak E10 3' and 5' splice sites as observed in other mRNAs (46, 52, 53). Further work is required to distinguish between the enhancer-dependent functions of SF2/ASF versus Tra2 and their interaction targets within the splicing complex.

Others have attempted to identify E10 trans-acting factors using templates that encompass most or all of E10 including the PPE (31, 47). N279K targets bound endogenous Tra2 in UV cross-linking assays and exogenously added Tra2 in affinity purification assays (31). In contrast to our results, Tra2 was also affinity-purified from targets with mutation 280K or with the entire nine-nucleotide PPE deleted. Another report showed increased immunoprecipitation of exogenous Tra2, SF2/ASF, and SRp30c with N279K targets, although significant associations were also detected with 280K (47). Because both studies used almost the entire E10 sequence as a template and because binding in the absence of UV cross-linking was observed with 280K and PPE deletion constructs, these experiments cannot distinguish between direct binding to the PPE, indirect binding through factors already bound to the PPE, and the presence of additional SF2/ASF and Tra2 E10-binding sites outside of the PPE. While this manuscript was in preparation, Wang et al. (56) recently reported that Tra2 overexpression enhances E10 splicing from N279K templates by antagonizing SRp55 and SRp30c associations with a regulatory sequence located immediately upstream of the PPE.

The cumulative interactions between multiple cis-elements and trans-factors result in balanced E10 splicing, which maintains a normal 4R/3R ratio of 1. The requirement for multiple ESEs in E10 and the intron splicing modulator in I10 reflects the need to overcome the presence of: weak splice sites, silencer sequences in E10 and I10, and large introns that flank E10. Our results provide a biochemical explanation for the opposite effects of mutations 280K and N279K on E10 splicing. The altered associations and distinct regulatory functions of SF2/ASF and Tra2 proteins may contribute to the different clinical phenotypes associated with these splicing mutations. The altered activity (phosphorylation pattern) (54, 55) or expression levels of SF2/ASF and Tra2 in different brain regions provide a pathogenic mechanism for abnormal 4R/3R tau isoform ratios observed not only in FTDP-17 but also in related disorders progressive supranuclear palsy, corticobasal degeneration, and Pick disease. SF2/ASF and Tra2 are thus modifiers of disease. Because tau is involved in over 22 neurodegenerative disorders including Alzheimer disease, revealing normal and aberrant mechanisms in tau expression will be valuable not only in understanding its pathogenic role in the central nervous system but also in the potential for designing RNA- or protein-based therapeutics.

	FOOTNOTES

 
^* This work was supported by an Alzheimer Disease research grant from the American Health Assistance Foundation (to I. D.) and by NIA, National Institutes of Health Grant RO1 AG11762 (to G. D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: VAPSHCS, GRECC/182B, 1660 S. Columbian Way, Seattle, WA 98108. Tel.: 206-277-1071; Fax: 206-764-2569; E-mail: iands{at}u.washington.edu.

² The abbreviations used are: E10, exon 10; ESE, exon splicing enhancer; FTDP-17, frontotemporal dementia with parkinsonism chromosome 17-type; I10, intron 10; PPE, polypurine enhancer; RRM, RNA recognition motif; RS, arginine/serine-rich protein interaction domain; siRNA, small interfering RNA; SR, Arg/Ser-rich splicing factors; 3R, three-repeat tau isoforms; 4R, four-repeat tau isoforms; RT, reverse transcription.

	ACKNOWLEDGMENTS

 
We thank Adrian Krainer for providing the SF2/ASF monoclonal antibody, William Mattox and Brunhilde Wirth for Tra2 expression vectors, as well as Alan Zahler and Mark McNally for helpful discussions regarding RNA affinity column assays. We also thank Zanna Schiffelbein and Leojean Anderson for technical help.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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