- Research article
- Open Access
Functional and cellular characterization of human Retinoic Acid Induced 1 (RAI1) mutations associated with Smith-Magenis Syndrome
- Paulina Carmona-Mora†1, 2,
- Carolina A Encina†2, 3,
- Cesar P Canales1, 2,
- Lei Cao1,
- Jessica Molina2,
- Pamela Kairath2, 3,
- Juan I Young1, 2, 4 and
- Katherina Walz1, 2Email author
© Carmona-Mora et al; licensee BioMed Central Ltd. 2010
- Received: 18 March 2010
- Accepted: 25 August 2010
- Published: 25 August 2010
Smith-Magenis Syndrome is a contiguous gene syndrome in which the dosage sensitive gene has been identified: the Retinoic Acid Induced 1 (RAI1). Little is known about the function of human RAI1.
We generated the full-length cDNA of the wild type protein and five mutated forms: RAI1-HA 2687delC, RAI1-HA 3103delC, RAI1 R960X, RAI1-HA Q1562R, and RAI1-HA S1808N. Four of them have been previously associated with SMS clinical phenotype. Molecular weight, subcellular localization and transcription factor activity of the wild type and mutant forms were studied by western blot, immunofluorescence and luciferase assays respectively. The wild type protein and the two missense mutations presented a higher molecular weight than expected, localized to the nucleus and activated transcription of a reporter gene. The frameshift mutations generated a truncated polypeptide with transcription factor activity but abnormal subcellular localization, and the same was true for the 1-960aa N-terminal half of RAI1. Two different C-terminal halves of the RAI1 protein (1038aa-end and 1229aa-end) were able to localize into the nucleus but had no transactivation activity.
Our results indicate that transcription factor activity and subcellular localization signals reside in two separate domains of the protein and both are essential for the correct functionality of RAI1. The pathogenic outcome of some of the mutated forms can be explained by the dissociation of these two domains.
- Transcription Factor Activity
- Nuclear Localization Signal
- Wild Type Protein
- Contiguous Gene Syndrome
- Dosage Sensitive Gene
A large fraction of genome variation between individuals is comprised of submicroscopic copy number variation of DNA segments (CNVs) [1–6]. Genomic disorders are the clinical manifestation of pathological CNV. They are frequent conditions (~1 per 1,000 births) and often sporadic resulting from de novo rearrangements . In a subset of such conditions the rearrangements comprise multiple unrelated contiguous genes that are physically linked and thus have been referred to as contiguous gene syndromes (CGS). An increasing number of CGS are being described, each of them presenting a complex and specific phenotype. Although several genes are usually present in the segmental aneuploidy; only a small subset of them conveys phenotypes as a function of copy number alteration. These particular genes are referred to as "dosage sensitive genes".
The Smith-Magenis Syndrome, SMS, (OMIM# 182290) is a CGS associated with a microdeletion within chromosome 17 band p11.2. SMS was first described in 1986 with a birth prevalence estimated at 1/25,000. The clinical phenotype includes craniofacial abnormalities, brachydactyly, self injurious behavior, sleep abnormalities and mental retardation. Less commonly reported is cleft palate, congenital heart defects, seizures, hearing impairment and urinary tract anomalies . Molecular studies revealed a common deleted region of ~4 Mb in the majority of SMS patients (>70-80%) [9–11]. Unusual sized deletions (smaller or larger) were observed in 20-25% of patients [12–14]. By examining the breakpoints in unusual sized deletions, the SMS critical region was redefined to a ~950 kb interval, in which 15 genes and eight predicted genes were present [11–14]. Retinoic Acid Induced 1 gene (RAI1) is located in the middle of the SMS critical region. Point mutations (nonsense and frameshift as well as missense alleles) in RAI1 were identified in patients with clinical presentation of SMS but no molecular deletion found by FISH [15–18], suggesting that RAI1 is the dosage sensitive gene causative of SMS. Studies on mouse models  and humans [20, 21] indicate that RAI1 is likely the dosage sensitive gene responsible for clinical features in the Potocki-Lupski Syndrome (PTLS), (OMIM# 610883) a neurobehavioral disorder with autistic features that is caused by reciprocal duplication of the 17p11.2 region [20–23]. The RAI1 gene consists of six exons  that span over 120 kb. The third exon contains >90% of the coding region and it is within this exon where all mutations have been identified to date.
Little is known about the cellular and developmental role of RAI1. Expression of GT1, a splice variant of Rai1 was markedly up-regulated by treatment with retinoic acid in a mouse carcinoma cell line P19 . A polymorphic CAG repeat is present in the N-terminus of the RAI1 protein, the length of which is associated with the age of onset of spinocerebellar ataxia type 2  and the response to neuroleptic medication in schizophrenia . Moreover, RAI1 was recently associated with non syndromic autism . Bioinformatic analyses have suggested that RAI1 might be a transcriptional regulator [15, 16]. RAI1 contains several patches of 50% similarity with TCF20, a transcriptional cofactor, and these two genes have a similar gene structure. RAI1, in both human and mouse, have two putative bipartite nuclear localization signals (NLSs) predicted in silico, and possesses a zinc finger like plant homeo domain (PHD) in the C-terminus, which is also present in the trithorax family of chromatin remodeling transcriptional regulators . In vitro studies of the murine Rai1 protein indicated that it can be transported to the nucleus and has transactivation activity . However, no systematic analysis of the human RAI1 protein has been done. Here we describe the characterization of the wild type RAI1 protein, plus five mutated forms of the protein, including four that have been associated with the SMS clinical phenotype. Subcellular localization of the resulting proteins and transcription factor activity were studied for the wild type and mutant forms. Our results indicate that the transcription factor activity and subcellular localization of the protein are essential for the pathogenic outcome of some of the mutated forms.
The transcription factor activity of the murine Rai1 protein is dependent on the cell type
Human RAI1 is a nuclear protein with transcription factor activity
In a first attempt to understand and characterize the function of human RAI1 we generated the full-length cDNA coding for the wild type protein. The full-length cDNA was obtained as described in Material and Methods. The final full-length sequence is the same as the one reported in NM_030665 with SNP A/G at position 1992. In order to have an easy way to identify the transfected RAI1 from the endogenous protein, an HA tag was added in the 3' end (figure 1A). Since we found that Rai1 activity was dependent of cellular type, we performed all our studies in mouse Neuro-2a neuroblastoma cells. Neuro-2a cell line was preferred over the SH-SY5Y (human neuroblastoma) cell line despite its murine origin, since the human ones grow at an extremely slow rate and making them very difficult to transfect. In order to determine the molecular weight of the protein, both clones the RAI1 and RAI1-HA cDNA were transiently transfected, and 48 h post-transfection the cell lysate was run in a 10% SDS-PAGE gel. The predicted in silico molecular weight for RAI1 is 203 kDa. As can be observed in figure 1B, the molecular weight of both proteins is ~260 kDa, and the addition of the HA tag is not inducing instability of the resulting protein. The difference between the molecular weight predicted and the obtained could be explained by post-translational modifications (PTM) for example glycosylations. We found several predicted glycosylation sites on the first amino-terminal half of RAI1 (analysis in NetOGlyc 3.1 server, EXPASY Tools, http://www.expasy.org) .
To prove if the human protein has transcription factor activity, we subcloned the RAI1 and RAI1-HA full-length cDNA into a GAL4-binding domain vector (pCMV-BD, Stratagene). It is important to note that the presence of the GAL4-binding domain is enough to localize the protein to the nucleus . Transfection of each clone plus the reporter gene was carried out and 48 h post-transfection the cells were lysed and the activity of the luciferase reporter gene was measured. As can be seen in figure 1D, the expression of the wild type RAI1 and RAI1-HA derives in an 67.5 +/- 31.8 and 58.8 +/- 11.1 fold increase of the luciferase activity respectively, indicating that the human protein presented transcription factor activity and that the addition of the HA tag did not interfere with the activity of the resulting protein. RAI1 has a bipartite nuclear localization signal predicted by informatics analysis . Nuclear subcellular localization was found for both, the RAI1 and RAI-HA proteins (figure 1C) at 36 h post-transfection and this result was confirmed with two different antibodies, one that recognized an epitope within the first 30 amino acids in the N-terminus portion of the RAI1 protein and the anti HA antibody, which recognized the C-terminus end in the RAI1-HA clone. These results indicate that the human RAI1 protein, with or without the HA tag, has a nuclear localization and the ability to activate the transcription of a reporter gene.
The N-terminal truncated RAI1 polypeptides retain the transcription factor activity but localize in the cytoplasm
We evaluated the transcription factor activity for the truncated products by subcloning the three mutant forms cDNA into the pCMV-BD vector and posterior transfection of each clone plus the reporter gene into Neuro-2a cells. Forty eight hours post-transfection the cells were lysed and the activity of the luciferase reporter gene was measured. As can be seen in figure 2C, all truncated proteins gave an increment of 233.2 +/- 19.2 (for RAI1 2687delC), 179.1 +/- 24.1 (for RAI1 3103delC), and 282.5 +/- 23.8 (for RAI1 R960X), in percentage of activation in comparison with the 100% of the wild type, indicating that the truncated polypeptides retained the transcription factor activity. All the truncated proteins showed a significant increased percentage of transcription activation when compared to the wild type protein (P value ≤ 0.01). The following step was to evaluate the subcellular localization of the truncated fragments in the absence of the GAL4-binding domain since the presence of this domain is enough to localize any protein to the nucleus . The resulting truncated polypeptides would not include the hypothetical Nuclear Localization Signals (NLSs). Cytoplasmatic subcellular localization was found for all the
RAI1-HA 2687delC, RAI1-HA 3103delC and RAI1 R960X polypeptides (figure 2D). All together these results indicate that RAI1-HA 2687delC, RAI1-HA 3103delC and RAI1 R960X are generating truncated polypeptides that retain the transcription factor activity. However, they are not localized in the nucleus but in the cytoplasm of cells.
Nuclear localization signals are situated in the C-terminus of the human RAI1 protein
No alterations were found for RAI1 Q1562R and RAI1 S1808N mutants
For both mutants we evaluated the transcription factor activity, in the same way that was previously done for the other proteins. As can be seen in figure 4C, both RAI1-HA Q1562R and RAI1-HA S1808N mutated proteins can give an increment of 120.5 +/- 20.4 and 113.7 +/- 8.6 percentage of activation of luciferase activity respectively, when compared with the wild type as 100%. This is similar to what was found for the wild type RAI1-HA protein, indicating that the mutated polypeptides retained the transcription factor activity. We next evaluated the subcellular localization of the mutated proteins, and nuclear localization was found for both of them (figure 4D). All together these results indicate that RAI1 Q1562R and RAI1 S1808N mutations are not affecting the stability, transcription factor activity or subcellular localization of the protein.
Alterations in the RAI1 gene have been associated with Smith-Magenis and Potocki-Lupski Syndromes whose clinical presentation includes autistic features, obsessive-compulsive behaviors, attention deficit, developmental delay, mental retardation, EEG abnormalities, sleep disturbances, self-injurious and maladaptive behaviors, among others [19–21]. In addition to this, RAI1 gene was associated with spinocerebellar ataxia (SCA2) , neuroleptic response in patients with schizophrenia , neurobehavioral traits manifested in SMS patients with RAI1 loss-of-function alleles [33–46] and non syndromic autism . Despite its relationship with these important traits, there is lack of information about the molecular function of human RAI1.
Several heterozygous nucleotide variations in RAI1 gene have been found in SMS patients without 17p11.2 deletions. These mutations include nonsense and missense mutations besides deletions of one or multiple nucleotides [15–18, 43].
Within the RAI1 gene coding sequence there are several mononucleotide repeats. Intriguingly all five single base pair frameshift mutations found in the RAI1 gene associated with the SMS phenotype were in the C-tracts although the number of G-tracts in the RAI1 coding region is comparable with the C-tracts . Interestingly, in our laboratory a spontaneous deletion of a C in a four C-tract at the position 2687 occurred, providing further evidence that polyC-tract may be a preferential target for frameshift mutations in RAI1.
Here we generated and studied the full-length cDNA of human wild type RAI1 and five mutated forms of the protein. Four of them previously associated with SMS. As deletions appear to be a very frequent event in the RAI1 coding region, we also included in this study the spontaneously generated 2687delC. In order to complement these studies, we have constructed two smaller proteins corresponding to the residues 1038-1906 and 1229-1906.
It was previously reported that mouse Rai1 has moderate transactivational activity in HeLa cells . Here we confirmed those previous findings and showed that Rai1 has a stronger transactivation activity in Neuro-2a cells, the neuronal derived cell line. These results suggest specificity in the transcription machinery that RAI1 could be part of and further indicate the importance of its role in neuronal derived tissues, since RAI1 is a dosage sensitive gene involved in neurobehavioral phenotypes.
Here we report that the wild type human RAI1 protein has transcription factor activity. Our studies of truncated proteins associated with 2687delC and 3103delC frameshifts plus R960X mutant, showed transcription factor activity significantly higher than the wild type protein for the resulting polypeptides. Moreover, the analysis of the two smaller proteins corresponding to the residues 1038-1906 and 1229-1906 showed no capability of activating transcription themselves. According to our results, the human RAI1 showed that the most important region of the protein for stimulating transcription is the N-terminal half, until residue 1034. From the residue 1038 to the end of RAI1 a fragment able to regulate the transactivational activity of the protein or the machinery associated may be present, since all truncated proteins showed a significant increase in transcription activity when compared to the full-length protein. In the N-terminal region, corresponding approximately to the first half of the protein, there is a homology of ~84% between human and murine RAI1. For the murine Rai1 protein, putative transactivation domains were mapped by fusion of different regions to GAL4-BD . The polypeptides containing residues 1-582 or encompassing residues 583 to 1142 have been shown to have transactivation activity while no transactivation activity was found in the region containing the remaining half; consistent with what is reported here for the human protein.
By bioinformatics analysis two putative bipartite nuclear localization signals (NLSs) were defined for RAI1 at positions 1113 and 1176 aa respectively . Despite that we cannot discard the participation of the previously described NLS in the nuclear localization of the RAI1 protein, our results indicate that there is a nuclear localization signal that resides between 1229 aa to the end since the two smaller proteins 1038-1906 and 1229-1906 were able to localize in the nucleus; while the two N-terminal polypeptides were retained in the cytoplasm. These results suggest the presence of another fragment within the C-terminal half of RAI1 that modulates the nuclear localization of the protein. Accordingly, in vitro studies of the murine Rai1 protein indicated that it can be transported to the nucleus and that the nuclear localization signals reside between the 1134-1164 and 1203-1229 aa. Besides, on the C-terminal half of Rai1 there are other regions that are also able to transport the protein to the nucleus .
The truncated proteins generated by mutations 2687delC, 3103delC and R960X showed to be functional and moreover, they have an increased capability for activating the transcription when compared to the wild type protein. However, none of the resulting polypeptides were able to localize in the nucleus, suggesting that this inability (even retaining transcription factor activity) would be the main pathological way of action for these mutations.
Finally, there are spare cases of SMS patients with mutations in the RAI1 gene. We generated two independent mutations, RAI1 Q1562R and RAI1 S1808N. No abnormal molecular weight, subcellular localization or transcription factor activity were found for any of these mutants, suggesting that they may be implicated in the association with other proteins or some other functional role of RAI1 that is not known to date.
We were able to demonstrate that the human wild type protein has transcription factor activity, and is able to localize to the nucleus. Moreover, we could define a transcription factor domain within the first 1034 aa and that the nuclear localization signals reside in the second half the protein. Altogether the analysis of the mutant proteins indicates that both halves are necessary for the correct subcellular localization and function of the protein, and that other molecular/functional role of RAI1 remains unknown.
The full-length clone of murine Rai1 was kindly provided by Dr. James Lupski, Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, Texas, USA. The sequence of influenza virus hemagglutinin epitope (HA) was added to the 3' extreme to generate a tag by PCR using pCR3.1 Rai1 as template, with the primers Forward: GCCTCATCCTGAGAAGCAAC and Reverse: AAGTCTAGATTAAGCGTAATCTGGAACATCGTATGGGTACAACGGCAGCCTCTTATGTTTG. The reverse primer contains the last 22 nucleotides of Rai1 cDNA without its stop codon, the coding sequence of hemagglutinin epitope, followed by a stop codon and an XbaI restriction site. The product of the reaction was purified and ligated into pGEM-T easy vector (Promega) and sequenced. In order to obtain the full-length Rai1-HA clone this PCR fragment was subcloned into the pCR3.1 Rai1 between the AccI and XbaI sites.
As a first step to obtain the complete RAI1 cDNA a PCR product was generated by amplification of a human brain cDNA library with the following primers: Forward: GGCCTGGTAAATGTGGGCACCGGG and Reverse: AAGGCGGCCGCTTAAGCGTAATCTGGAACATCGTATGGGTACGGCAGCCTCTTATGTTTGGGAC. The reverse primer was designed to add the influenza virus hemagglutinin epitope tag (HA) and the NotI restriction site at the 3' extreme. Finally, the partial 5' end cDNA RAI1 clone (KIAA1820, Kazusa DNA Research Institute, Japan) was ligated with the ~1.5 kb fragment that contained the 3' end of the cDNA with BamHI and NotI enzymes. The complete RAI1 cDNA clone was totally sequenced.
Three different point mutants were generated by site-directed mutagenesis by PCR with the kit QuickChange Site-Directed Mutagenesis XL Kit (Stratagene) utilizing the following primers: for the mutation A4685G, Forward: GCGACGACGACGGCAGCAGGTGCTG and Reverse: CAGCACCTGCTGCCGTCGTCGTCGC; for the mutation G5423A, Forward: CAAACATGAGTGCAACAAGGAGGCTC and Reverse: GAGCCTCCTTGTTGCACTCATGTTTG, for the deletion 3103delC, Forward: CTGCACAGGGCCCCCCAGGGACAGATGGAA and Reverse: CTTCCATCTGTCCCTGGGGGGCCCTGTGCAG. The deletion 2687delC was a spontaneous deletion found during the sequencing. The cDNA for the truncated protein R960X was generated by PCR with the following primers, Forward: CTGACCGGTGGCTGGAGGAC and Reverse: TGGTGGAATCCCCTGGAGCTCCTACTCC. The product contains a stop codon instead of base 2878. This fragment was subcloned into full-length RAI1 cDNA with the enzyme SacI. All clones were verified by DNA sequencing.
Two C-terminal fragments of the RAI1 protein were generated using the following primers: for the fragment 1038-1915, Forward: GGGACAGATGGAAGGGGCTGG and for the fragment 1229-1915, Forward: GTCGGCCTTCATGGCGCCG. Both amplification reactions were performed with the same primer reverse used for generating the full-length clone, detailed above, which contains the HA tag and the NotI restriction site at the 3' extreme. PCR products obtained were fully verified by DNA sequencing.
For expression analysis, the cDNAs of RAI1 wild type and mutant forms were subcloned in pALTER-MAX vector (Promega).
For KIAA1820 [GenBank: AB058723], Mouse Rai1 [GenBank: NM_009021 and Swiss-Prot: Q61818]. For Human RAI1 [GenBank: NM_030665 and Swiss-Prot: Q7Z5J4].
Neuro-2a and HeLa cells were grown in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 μg/ml) (Gibco by Invitrogen) at 37°C with 5% CO2 until 95% confluence was attained.
Immunofluorescence and Western blot analysis
To study the expression of the proteins generated, Neuro-2a cells were transfected using Lipofectamine 2000 (Invitrogen), with the mouse plasmid pCR3.1 Rai1-HA and human constructs pALTER-MAX RAI1-HA wild type, RAI1-HA Q1562R, RAI1-HA S1808N, RAI1 R960X, RAI1-HA 2687delC, RAI1HA 3103delC, RAI1HA 1038-1915 and RAI1HA 1229-1915. All transfections were performed according to manufacturer's protocol.
For immunofluorescence, cells were fixed 36 h after transfection with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 in PBS. Subcellular localization of RAI1-HA wild type and mutant forms of the protein were detected using rabbit anti human RAI1 polyclonal antibody (1:200, ab58658 Abcam) whose epitope locates at the amino-terminal of RAI1. A secondary antibody conjugated to Alexa fluor 488 (1:500) was used. Cells were mounted in a medium with DAPI (Vector Laboratories).
For western blot analysis, total protein extracts were prepared 48 h after transfection. Cells were lysed in 100 μl of protein extraction and loading buffer (2% SDS, 2 M Urea, 10% Glycerol, 10 mM Tris pH 6.8, 0.002% Bromophenol Blue and 10 mM DTT) plus 1:200 protease inhibitor cocktail (SIGMA). The samples were homogenized passing 20 times through a syringe and warmed up to 95°C for 5 minutes. 25 μl of each cell lysate was loaded onto 10% SDS-polyacrylamide gels with Tris/glycine running buffer and transferred to a 0.2 μm polyvinylidene fluoride (PVDF, Bio-Rad) membrane. Immunodetection was performed using rabbit anti human RAI1 polyclonal antibody (1:1000, Abcam) and rabbit anti β-tubulin (1:1000, sc-9104 Santa Cruz). Results were visualized by chemiluminiscence.
The detection of murine Rai1-HA and proteins 1038-1915 and 1229-1915 was done with anti HA high affinity antibody (1:5000 for Western blot and 1:1000 for immunofluorescence clone 3F10, Roche).
Reporter gene assays
Transient transfections in Neuro-2a cells were performed in 35 mm plates. The amounts of plasmid DNA used were according to manufacturer's protocol. GAL4-BD fusions of mouse and human RAI1 wild type and all of its mutant forms were co-transfected with the luciferase reporter plasmid pFR-Luc (Stratagene) and the β-Galactosidase expression plasmid pSV-β-Galactosidase (for normalization of the transfection efficiency). Forty eight hours post-transfection, cells were lysed and luciferase assays were performed with Luciferase Assay Kit (Stratagene), according to manufacturer's instructions. The Relative Lights Units (RLUs) were measured in duplicate in a Syrius Luminometer (Berthold Detection Systems).
β-Galactosidase activities were measured with 810 μl of β-Galactosidase buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgCl2 and 50 mM β-mercaptoethanol) for 50 μl of cell lysates plus 160 μl ONPG reagent (60 mM Na2HPO4,40 mM NaH2PO4 and 4 mg/ml ONPG (o-Nitrophenyl-β-D-Galactopyranoside). Incubations were performed at 30°C, reactions were stopped by adding 250 μl of Na2CO3 1 M. The absorbance was read at 420 nm. Each cell assay was carried out in duplicate.
We thank Ana Lorenzo and Irene Perez for administrative support and English editing of the manuscript. This work was supported by FONDECYT [grant 1061067 (KW), 1051079 (JIY)], FIC [R03 TW07536 (KW)]. The Centro de Estudios Científicos (CECS) is funded by the Chilean Government through the Millennium Science Initiative and the Centers of Excellence Base Financing Program of Conicyt. CECS is also supported by a group of private companies which at present include Antofagasta Minerals, Arauco, Empresas CMPC, Indura, Naviera Ultragas and Telefónica del Sur.
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