Regulation of neuron-specific alternative splicing of neurofibromatosis type 1 pre-mRNA

Abstract

Neurofibromatosis type 1 (NF1) is one of the most common heritable autosomal dominant disorders. Alternative splicing modulates the function of neurofibromin, the NF1 gene product, by inserting the in-frame exon 23a into the region of NF1 mRNA that encodes the GTPase-activating protein-related domain. This insertion, which is predominantly skipped in neurons, reduces the ability of neurofibromin to regulate Ras by 10-fold. Here, we report that the neuron-specific Hu proteins control the production of the short protein isoform by suppressing inclusion of NF1 exon 23a, while TIA-1/TIAR proteins promote inclusion of this exon. We identify two binding sites for Hu proteins, located upstream and downstream of the regulated exon, and provide biochemical evidence that Hu proteins specifically block exon definition by preventing binding of essential splicing factors. In vitro analyses using nuclear extracts show that at the downstream site, Hu proteins prevent binding of U1 and U6 snRNPs to the 5' splice site, while TIAR increases binding. Hu proteins also decrease U2AF binding at the 3' splice site located upstream of exon 23a. In addition to providing the first mechanistic insight into tissue-specific control of NF1 splicing, these studies establish a novel strategy whereby Hu proteins regulate RNA processing.

FIG. 1.

Hu proteins suppress inclusion of NF1 exon 23a. (a) Diagram depicting the alternative inclusion of exon 23a in the NF1 pre-mRNA. The 63-nucleotide (nt) in-frame exon 23a is specifically excluded in neurons. The arrows indicate the oligonucleotides used to analyze endogenous NF1 splicing by RT-PCR. The sequence of exon 23a and its surrounding introns is shown, with the exon sequence capitalized. AU-rich sequences are underlined. (b) Association of Hu proteins and the NF1 pre-mRNA in CA77 cells. Nuclear extract prepared from CA77 cells was immunoprecipitated with anti-Hu sera. RNA was isolated from either total nuclear extract or immunoprecipitated pellet and analyzed by RT-PCR. Lanes without RT (−) are included as controls. The oligonucleotides used to amplify the NF1 pre-mRNA anneal to sequences in introns upstream and downstream of exon 23a, and the resulting PCR product is 250 nucleotides, while the oligonucleotides used to amplify the GAPDH pre-mRNA anneal to sequences in intron 3 and exon 5, and the resulting PCR product is 585 nucleotides. (c) Correlation of skipping of exon 23a and Hu protein expression. Inclusion of exon 23a in the endogenous NF1 pre-mRNA in HeLa and CA77 cells was detected by RT-PCR. Amplification bands resulting from exon 23a inclusion (267 nucleotides) or exclusion (204 nucleotides) are indicated. Expression of neuron-specific Hu proteins in the two cell lines was detected by Western blot analysis using anti-Hu patient sera. (d) RT-PCR analysis of the NF1 reporter pre-mRNA. HeLa or CA77 cells were cotransfected with the NF1 reporter construct shown in the top panel and increasing amounts (0.5 and 1 μg) of PTB or mHuC. RT-PCR was carried out using total RNA isolated from the transfected cells and the oligonucleotides indicated in the diagram. Amplification products resulting from exon 23a inclusion (309 nucleotides) or exclusion (246 nucleotides) are indicated. The percentage of NF1 exon 23a inclusion is displayed in the bar graphs. The expression of transfected PTB or mHuC in HeLa cells was verified by Western blot analysis. Error bars indicate standard deviations.

FIG. 2.

Overexpression of Hu proteins in HeLa and PC12 cells reduces inclusion of the endogenous NF1 exon 23a. (a) Transient transfection of PTB or mHuC in HeLa cells. Inclusion of endogenous NF1 exon 23a was detected by RT-PCR using total RNA isolated from the transfected cells and the oligonucleotides shown in Fig. 1a. The level of overexpressed proteins was detected by Western blot analysis. (b) Stable transfection of PTB or mHuC in PC12 cells. Inclusion of endogenous NF1 exon 23a was detected by RT-PCR. Error bars indicate standard deviations.

FIG. 3.

Two AU-rich sequence elements flanking exon 23a are important for regulated splicing of NF1 exon 23a. (a) A diagram indicating mutated AU-rich sequences in the reporter. nt, nucleotides. (b) Left, the wild-type or mutant (mut) reporters were cotransfected with empty vector or mHuC cDNA in HeLa cells. Inclusion of exon 23a from the NF1 pre-mRNA was detected by RT-PCR. A Western blot showing expression of the Xpress-tagged mHuC is included. U170K protein is shown as a loading control for the Western blot analysis. Right, the wild-type or mutant reporters were transfected into CA77 cells. Error bars indicate standard deviations. dn, down; dl, double.

FIG. 4.

TIA-1/TIAR proteins promote inclusion of exon 23a. (a) Overexpression of TIAR in HeLa cells increases exon 23a inclusion. HeLa cells were cotransfected with the wild-type (left panel) or mutant (see Fig. 3a for mutant sequence) (middle panel) NF1 reporter construct and increasing amounts (0.5 and 1 μg) of TIAR. RT-PCR was carried out using total RNA isolated from the transfected cells and the oligonucleotides indicated in the diagram in Fig. 1. The percentage of NF1 exon 23a inclusion is displayed in the bar graphs. The expression of transfected TIAR was verified by Western blot analysis. Overexpression of TIAR in CA77 cells increases exon 23a inclusion (right panel). (b) siRNA knockdown of TIA-1 and TIAR in HeLa cells reduces inclusion of exon 23a. HeLa cells were transfected with a control siRNA (250 pmol), an siRNA against TIA-1 (125 pmol TIA-1 plus 125 pmol control siRNA), an siRNA against TIAR (125 pmol TIAR plus 125 pmol control siRNA), or siRNAs against both TIA-1 and TIAR (125 pmol TIA-1 plus 125 pmol TIAR siRNA). Results of RT-PCR and Western blot analysis are shown. U170K protein served as a loading control for the Western blot analysis. WT, wild type. Error bars indicate standard deviations.

FIG. 5.

The AU-rich sequence element immediately downstream of exon 23a is important for regulated inclusion of NF1 exon 23a by TIA-1/TIAR and Hu proteins. (a) A shorter NF1 reporter that contains only 75 nucleotides of the upstream intron shown in the diagram was cotransfected with TIAR or mHuC protein in HeLa cells. Inclusion of exon 23a from this short reporter was analyzed by RT-PCR. (b) A short reporter carrying the same downstream mutations in AU-rich sequence as shown in Fig. 3a was cotransfected with TIAR or mHuC protein. Inclusion of exon 23a from this mutant short reporter was analyzed by RT-PCR. Error bars indicate standard deviations.

FIG. 6.

TIA-1/TIAR and Hu proteins compete with each other in regulating inclusion of the NF1 exon 23a. (a) Binding of TIA and Hu proteins to the wild-type (WT) or mutant RNA substrate was detected by a UV cross-linking/IP assay using an in vitro-transcribed RNA containing exon 23a and 64 nucleotides (nt) of the upstream and 110 nucleotides of the downstream intron sequences (see diagram) and HeLa or CA77 nuclear extract. GST-mHuB (1 μg) was added to HeLa nuclear extract to detect the binding of Hu proteins to NF1 RNA substrate. Anti-TIA-1/TIAR or anti-Hu serum was used in this IP experiment. (b) RNA GST pull-down assay using either wild-type or mutant in vitro NF1 RNA substrate and HeLa nuclear extract. (c) Competition between Hu and TIA-1/TIAR proteins was examined by UV cross-linking/IP using HeLa nuclear extract supplemented with increasing amounts (0.1, 0.5, and 2.5 μg) of GST-mHuB protein. Anti-TIA-1/TIAR antibody and anti-Hu sera were both included in the IP step. (d) In vivo competition of TIAR and Hu proteins. The NF1 reporter (HMT-NF1 863/499) was cotransfected with a control vector plasmid, increasing amounts of TIAR expression plasmid (1 and 2 μg) (lane 2 and lane 3), or 2 μg of TIAR plasmid together with increasing amount of mHuC (1 and 2 μg) (lane 4 and lane 5) in CA77 cells. Inclusion of the NF1 exon 23a was detected by RT-PCR. (e) siRNA knockdown of HuC in CA77 cells. CA77 cells were cotransfected with the NF1 reporter (HMT-NF1 863/499) in the absence of siRNA (lane 1) or with increasing concentrations (150 pmol and 300 pmol) of a control siRNA (lanes 2 and 3) or HuC siRNA (lanes 4 and 5). Inclusion of the endogenously expressed NF1 exon 23a was detected by RT-PCR (left panel). The same siRNAs were cotransfected with mHuC expression plasmid in HeLa cells, and a Western blot of the total protein lysate isolated from the transfected cells is shown in the right panel. Error bars indicate standard deviations.

FIG. 7.

TIAR and Hu proteins regulate binding of U1 and U6 snRNA to the 5′ splice site of NF1 exon 23a. (a) Psoralen cross-linking of the NF1 RNA substrate indicated in Fig. 6a in the presence (lane 2) or absence (lane 1) of HeLa nuclear extract (N.E.). (b) Psoralen cross-linking of the NF1 RNA substrate in HeLa nuclear extract without added protein (lane 1) or with with increasing amounts (0.5 and 2.0 μg) of GST (lane 2 and lane 3), GST-TIAR (lanes 4 to 8), GST-mHuB (lane 9 and lane 10), or a truncated Hu protein, GST-mHuB RRM1,2 (lane 11 and lane 12). A 2′-O′-methyl ribo-oligonucleotide that specifically blocks the pre-mRNA binding region of U1, U2, or U6 snRNA was included to identify the cross-linked species (lanes 6 to 8). (c) Psoralen cross-linking of NF1 RNA substrate in HeLa nuclear extract, in which the endogenous TIA-1 and TIAR proteins were removed by immunodepletion. Lane 1, mock-depleted nuclear extract; lane 2, TIA-1/TIAR-depleted nuclear extract; lane 3, TIA-1/TIAR-depleted nuclear extract supplemented with 1 μg GST-TIAR. The TIA-1/TIAR protein levels in the mock- and TIA-1/TIAR-depleted nuclear extracts were examined by Western blotting. (d) Psoralen cross-linking of NF1 RNA substrate in HeLa nuclear extract (lane 1) and CA77 nuclear extract (lane 2). A Minx RNA substrate that contains a 5′ splice site was used as a control in this experiment (lanes 3 and 4). The levels of TIA-1/TIAR proteins and a U1 snRNP protein were detected by Western blotting. U170K protein was used to indicate the levels of U1 snRNPs in these nuclear extracts. (e) Psoralen cross-liking of the NF1 RNA substrate in HeLa nuclear extract supplemented with increasing amounts (0.5 and 4 μg) of GST-HuR (lane 2 and lane 3), GST-mHuC (lane 4 and lane 5), or GST-HuD (lane 6 and lane 7).

FIG. 8.

U2AF65 cross-linking with the RNA substrate indicated in the diagram. The ³²P-labeled, in vitro-transcribed RNA transcripts were UV cross-linked in HeLa cell nuclear extract in the presence of increasing amounts of buffer alone or of GST-mHuB RRM1,2 or mHuB (50 and 200 ng) and immunoprecipitated with the anti-U2AF65 antibody. nt, nucleotides.

FIG. 9.

A proposed model indicating how Hu proteins may regulate the neuron-specific exclusion of NF1 exon 23a.