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. 2012;7(9):e45155.
doi: 10.1371/journal.pone.0045155. Epub 2012 Sep 18.

RAI1 transcription factor activity is impaired in mutants associated with Smith-Magenis Syndrome

Paulina Carmona-Mora  1 , Cesar P Canales, Lei Cao, Irene C Perez, Anand K Srivastava, Juan I Young, Katherina Walz
Affiliations

RAI1 transcription factor activity is impaired in mutants associated with Smith-Magenis Syndrome

Paulina Carmona-Mora et al. PLoS One. 2012.

Abstract

Smith-Magenis Syndrome (SMS) is a complex genomic disorder mostly caused by the haploinsufficiency of the Retinoic Acid Induced 1 gene (RAI1), located in the chromosomal region 17p11.2. In a subset of SMS patients, heterozygous mutations in RAI1 are found. Here we investigate the molecular properties of these mutated forms and their relationship with the resulting phenotype. We compared the clinical phenotype of SMS patients carrying a mutation in RAI1 coding region either in the N-terminal or the C-terminal half of the protein and no significant differences were found. In order to study the molecular mechanism related to these two groups of RAI1 mutations first we analyzed those mutations that result in the truncated protein corresponding to the N-terminal half of RAI1 finding that they have cytoplasmic localization (in contrast to full length RAI1) and no ability to activate the transcription through an endogenous _target: the BDNF enhancer. Similar results were found in lymphoblastoid cells derived from a SMS patient carrying RAI1 c.3103insC, where both mutant and wild type products of RAI1 were detected. The wild type form of RAI1 was found in the chromatin bound and nuclear matrix subcellular fractions while the mutant product was mainly cytoplasmic. In addition, missense mutations at the C-terminal half of RAI1 presented a correct nuclear localization but no activation of the endogenous _target. Our results showed for the first time a correlation between RAI1 mutations and abnormal protein function plus they suggest that a reduction of total RAI1 transcription factor activity is at the heart of the SMS clinical presentation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Schematic representation RAI1 protein structure and point mutations associated with the SMS phenotype. A)
The structure of isoform 1 of RAI1 protein is schematically represented and the following domains are indicated according to their amino acidic position: polyglutamine tract (PolyQ), two different polyserine tracts (PolyS), a plant homeo domain PHD and bipartite nuclear localization signals, NLSs. Twenty SMS patients harboring a RAI1 point mutation are depicted: two nonsense, five missense and eleven frameshift mutations (including four patients with Q1035X). The two groups of mutations are represented. The mutations analyzed in the present study are shown in bold. B) Clinical features previously described as significantly different between SMS patients harboring a common, large, small or atypical deletion or RAI1 point mutation are listed. Four sets of comparisons are shown: 1. between SMS patients harboring a deletion versus RAI1 point mutation (groups I and II), 2. between SMS patients harboring a deletion versus RAI1 point mutation in the N-terminal half of the protein (groups I and 1), 3. between SMS patients harboring a deletion versus RAI1 point mutation in the C-terminal half (groups I and 2), 4. between SMS patients harboring a RAI1 point mutation in the N-terminal half versus the C-terminal half (groups 1 and 2). The significant differences found in each set of comparisons are represented with an asterisk (* = p<0.05). a: short stature (<5th percentile); b: Obese classification based on Body Mass Index (BMI): BMI >95th percentile for children and adolescents; BMI>30 for adults. N/A = not available.
Figure 2
Figure 2. Molecular and cellular evaluation of the p.1035fsX30 truncated protein. A)
Schematic representation of the RAI1 protein structure, which includes the polyglutamine tract (PolyQ, blue), polyserine tract (PolyS, green), bipartite nuclear localization signal (NLS, black), and the plant homeo domain (PHD, gray). The HA epitope (red) was added by PCR at the 3′ end of full length cDNA of RAI1 isoform 1. The p.1035fsX30 truncated protein is represented below the isoform 1 of RAI1. B) Neuro-2a cells were transfected either with the clones RAI1-HA wild type or RAI1 c.3103insC (p.1035fsX30). A Western blot using an antibody against RAI1 (α RAI1) was performed to calculate the molecular weight of RAI1 p.1035fsX30. The anti β-tubulin antibody (α β-tubulin) was used as loading control. C) The subcellular localization of the truncated protein was assessed by immunofluorescence using anti RAI1 (α RAI1). Neuro-2a cells were transfected with the plasmids coding for RAI1 wild type and RAI1 p.1035fsX30. As negative control, untransfected cells are also shown. Nuclei were stained with DAPI. The table indicates the subcellular localization N; nuclear, C; cytoplasmatic, of 200 cells, *: p≤0.05). D) Schematic representation of Neuro-2a cells that were co-transfected with RAI1 wild type or RAI1 c.3103insC fused with GAL4-BD (pCMV-BD), plus the luciferase reporter plasmid. β-galactosidase activity was used for normalization due to differences in transfection efficiency. E) Luciferase expression was utilized as a reporter for transactivation activity of RAI1. The wild type activity was considered as 100%. Values represent mean ± SEM. (RAI1-HA n = 9, RAI1 p.1035fsX30 n = 3; *: p≤0.05).
Figure 3
Figure 3. Evaluation of RAI1 expression in lymphoblastoid cells.
A) Western Blot for RAI1 expression in lymphoblastoid cells from patient BAB1852 and normal control. B) A band densitometric analysis of RAI1 isoforms was performed, and the percentages compared to the isoform 1 in cells from the control are shown. The band of ∼170 kDa found in patient cells represents truncated RAI1 isoform 1 and RAI1 isoform 4 proteins as a result of their similar molecular weight. (n = 4 for both samples, * p = 0.02; ** p≤1.78*10−6). Subcellular localization of RAI1 in C) control or D) BAB1852 lymphoblastoid cells. Whole Cell (WC), Cytoplasmic (C), Nuclear soluble (N), Chromatin Bound (CB) and Nuclear Matrix (NM) fractions were obtained from control or BAB1852 lymphoblastoid cells. RAI1 isoform 1 is mainly associated with chromatin and nuclear matrix. The antibodies Acetyl-histone H3 antibody (α Acetyl-histone H3, chromatin bound protein marker) and beta-tubulin antibody (α β-tubulin, cytoplasmic soluble fraction marker) were used as controls of the protocol for cell fractionation.
Figure 4
Figure 4. BDNF enhancer transactivation activity for two groups of RAI1 mutations. A)
Schematic representation of the plasmid construct used to measure the transactivation activity of RAI1 wild type or the mutants forms driven by the BDNF enhancer in HEK293T cells. The intronic sequence for BDNF gene enhancer region was amplified by PCR and added upstream of the SV40 promoter. B) HEK293T cells were co-transfected with a BDNF fused luciferase reporter plasmid, a β-galactosidase reporter plasmid, and either RAI1 isoform 1, p.1035fsX28, p.1035fsX30 or RAI1 R960X. Forty eight hours post-transfection the reporter proteins were measured from the cell lysates. Activation of the reporter for empty vector along with BDNF fused luciferase reporter plasmid and a β-galactosidase reporter plasmid (basal endogenous BDNF level) was used for normalization. (p≤0.002). RAI1 isoform 1 n = 9, RAI1 p.1035fsX28 n = 6, RAI1 p.1035fsX30 n = 9, RAI1 R960X n = 6. Values represent mean ± SEM. (* = p<0.05). C) The same experiment was performed for group 2 of point mutations mapping at the C-terminal half of RAI1. All the proteins analyzed exhibit impaired activation of the endogenous _target BDNF. RAI1 isoform 1 n = 3, RAI1 p.R1217Q n = 4, RAI1 p.Q1389R n = 4, RAI1 p.Q1562R n = 6 and RAI1 p.S1808N n = 9. Values represent mean ± SEM. (* = p<0.05.).
Figure 5
Figure 5. Schematic representation of RAI1 functional domains.
The schematic representation of all the mutants analyzed in this study is shown. An asterisk represents the missense mutations. The N and C-terminal halves of the protein are depicted with the description of their functional role.
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References

    1. Greenberg F, Lewis RA, Potocki L, Glaze D, Parke J, et al. (1996) Multi-disciplinary clinical study of Smith-Magenis syndrome (deletion 17p11.2). Am J Med Genet 62: 247–254. - PubMed
    1. Laje G, Morse R, Richter W, Ball J, Pao M, et al. (2010) Autism spectrum features in Smith-Magenis syndrome. Am J Med Genet C Semin Med Genet 154C: 456–462. - PMC - PubMed
    1. Slager RE, Newton TL, Vlangos CN, Finucane B, Elsea SH (2003) Mutations in RAI1 associated with Smith-Magenis syndrome. Nat Genet 33: 466–468. - PubMed
    1. Bi W, Saifi GM, Shaw CJ, Walz K, Fonseca P, et al. (2004) Mutations of RAI1, a PHD-containing protein, in nondeletion patients with Smith-Magenis syndrome. Hum Genet 115: 515–524. - PubMed
    1. Girirajan S, Elsas LJ 2nd, Devriendt K, Elsea SH (2005) RAI1 variations in Smith-Magenis syndrome patients without 17p11.2 deletions. J Med Genet 42: 820–828. - PMC - PubMed

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This work was supported in part by Le Jerome Foundation (KW). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.
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