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Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF.

Chen Z, Ma X, Zhang J, Hu J, Gorczynski RM - Nucleic Acids Res. (2010)

Bottom Line: A truncated CD200 (CD200(tr)) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200.Deletion or mutation of the ESE site decreased expression of the full-length CD200.Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200.

View Article: PubMed Central - PubMed

Affiliation: Transplant Research Division, Department of Surgery and Immunology, The Toronto Hospital, University of Toronto, Toronto, Canada. zhiqi.chen@utoronto.ca

ABSTRACT
CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200(tr)) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200.

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Direct binding of SF2/ASF to the ESE in exon 2 of CD200. A recombinant SF2/ASF (ΔRS domain) was incubated with a radiolabeled RNA oligonucleotide probe containing the ESE in exon 2 of CD200 (lane 1), with a radiolabeled RNA oligonucleotide plus 100× cold probe containing the same ESE (lane 2), or with a mutated ESE (lane 3). Lane 4 represents a radiolabeled RNA oligonucleotide only. The sequences of the RNA oligonucleotides are shown in ‘Materials and methods’ section. Free RNA probes and RNA–protein complexes were resolved by 5% nondenaturing polyacrylamide gel. The figure shown is representative of three independent experiments with similar results.
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Figure 5: Direct binding of SF2/ASF to the ESE in exon 2 of CD200. A recombinant SF2/ASF (ΔRS domain) was incubated with a radiolabeled RNA oligonucleotide probe containing the ESE in exon 2 of CD200 (lane 1), with a radiolabeled RNA oligonucleotide plus 100× cold probe containing the same ESE (lane 2), or with a mutated ESE (lane 3). Lane 4 represents a radiolabeled RNA oligonucleotide only. The sequences of the RNA oligonucleotides are shown in ‘Materials and methods’ section. Free RNA probes and RNA–protein complexes were resolved by 5% nondenaturing polyacrylamide gel. The figure shown is representative of three independent experiments with similar results.

Mentions: Since the ESE described above is known to contain a putative binding site for SF2/ASF, we investigated whether SF2/ASF binds to the ESE. An RNA–EMSA was performed. As shown in Figure 5, an RNA–protein complex was detected after the SF2/ASF recombinant protein with ΔRS domain was mixed with a radiolabeled RNA oligonucleotide containing the ESE site. This protein/RNA interaction is specific since SF2/ASF did not bind to a radiolabeled RNA oligonucleotide containing mutated ESE site and the above binding was eliminated by competing 100× unlabelled oligonucleotide containing the same ESE (Figure 5). Moreover, this binding was not competed by the same level of cold oligonucleotide with the ESE site mutated (data not shown).Figure 5.


Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF.

Chen Z, Ma X, Zhang J, Hu J, Gorczynski RM - Nucleic Acids Res. (2010)

Direct binding of SF2/ASF to the ESE in exon 2 of CD200. A recombinant SF2/ASF (ΔRS domain) was incubated with a radiolabeled RNA oligonucleotide probe containing the ESE in exon 2 of CD200 (lane 1), with a radiolabeled RNA oligonucleotide plus 100× cold probe containing the same ESE (lane 2), or with a mutated ESE (lane 3). Lane 4 represents a radiolabeled RNA oligonucleotide only. The sequences of the RNA oligonucleotides are shown in ‘Materials and methods’ section. Free RNA probes and RNA–protein complexes were resolved by 5% nondenaturing polyacrylamide gel. The figure shown is representative of three independent experiments with similar results.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2965252&req=5

Figure 5: Direct binding of SF2/ASF to the ESE in exon 2 of CD200. A recombinant SF2/ASF (ΔRS domain) was incubated with a radiolabeled RNA oligonucleotide probe containing the ESE in exon 2 of CD200 (lane 1), with a radiolabeled RNA oligonucleotide plus 100× cold probe containing the same ESE (lane 2), or with a mutated ESE (lane 3). Lane 4 represents a radiolabeled RNA oligonucleotide only. The sequences of the RNA oligonucleotides are shown in ‘Materials and methods’ section. Free RNA probes and RNA–protein complexes were resolved by 5% nondenaturing polyacrylamide gel. The figure shown is representative of three independent experiments with similar results.
Mentions: Since the ESE described above is known to contain a putative binding site for SF2/ASF, we investigated whether SF2/ASF binds to the ESE. An RNA–EMSA was performed. As shown in Figure 5, an RNA–protein complex was detected after the SF2/ASF recombinant protein with ΔRS domain was mixed with a radiolabeled RNA oligonucleotide containing the ESE site. This protein/RNA interaction is specific since SF2/ASF did not bind to a radiolabeled RNA oligonucleotide containing mutated ESE site and the above binding was eliminated by competing 100× unlabelled oligonucleotide containing the same ESE (Figure 5). Moreover, this binding was not competed by the same level of cold oligonucleotide with the ESE site mutated (data not shown).Figure 5.

Bottom Line: A truncated CD200 (CD200(tr)) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200.Deletion or mutation of the ESE site decreased expression of the full-length CD200.Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200.

View Article: PubMed Central - PubMed

Affiliation: Transplant Research Division, Department of Surgery and Immunology, The Toronto Hospital, University of Toronto, Toronto, Canada. zhiqi.chen@utoronto.ca

ABSTRACT
CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200(tr)) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200.

Show MeSH
Related in: MedlinePlus