Abstract
BackgroundSmith-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.ResultsWe 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.ConclusionOur 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.
Highlights
Smith-Magenis Syndrome is a contiguous gene syndrome in which the dosage sensitive gene has been identified: the Retinoic Acid Induced 1 (RAI1)
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 results showed that the transcription factor activity in HeLa cells is 2.2 +/- 1.5 folds over the empty vector and in Neuro-2a cells is 61.3 +/- 11.2 indicating that the murine protein has a transactivational activity several times stronger in the Neuro-2a cell line than in HeLa cells, suggesting that there is a specific machinery in neuronal cells that may be related to Rai1 transcription factor activity
Summary
Smith-Magenis Syndrome is a contiguous gene syndrome in which the dosage sensitive gene has been identified: the Retinoic Acid Induced 1 (RAI1). A large fraction of genome variation between individuals is comprised of submicroscopic copy number variation of DNA segments (CNVs) [1,2,3,4,5,6]. 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 [7]. In a subset of such conditions the rearrangements comprise multiple unrelated contiguous genes that are physically linked and 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. Several genes are usually present in the segmental aneuploidy; only a
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