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Characterization of novel isoforms and evaluation of SNF2L/SMARCA1 as a candidate gene for X-linked mental retardation in 12 families linked to Xq25-26.

Lazzaro MA, Todd MA, Lavigne P, Vallee D, De Maria A, Picketts DJ - BMC Med. Genet. (2008)

Bottom Line: Our results demonstrate that there are numerous splice variants of SNF2L that are expressed in multiple cell types and which alter subcellular localization and function.SNF2L mutations are not a cause of XLMR in our cohort of patients, although we cannot exclude the possibility that regulatory mutations might exist.Nonetheless, SNF2L remains a candidate for XLMR localized to Xq25-26, including the Shashi XLMR syndrome.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ottawa Health Research Institute, 501 Smyth Road, Ottawa, ON K1H 8L6, Canada. maribeth_lazzaro@hc-sc.gc.ca

ABSTRACT

Background: Mutations in genes whose products modify chromatin structure have been recognized as a cause of X-linked mental retardation (XLMR). These genes encode proteins that regulate DNA methylation (MeCP2), modify histones (RSK2 and JARID1C), and remodel nucleosomes through ATP hydrolysis (ATRX). Thus, genes encoding other chromatin modifying proteins should also be considered as disease candidate genes. In this work, we have characterized the SNF2L gene, encoding an ATP-dependent chromatin remodeling protein of the ISWI family, and sequenced the gene in patients from 12 XLMR families linked to Xq25-26.

Methods: We used an in silico and RT-PCR approach to fully characterize specific SNF2L isoforms. Mutation screening was performed in 12 patients from individual families with syndromic or non-syndromic XLMR. We sequenced each of the 25 exons encompassing the entire coding region, complete 5' and 3' untranslated regions, and consensus splice-sites.

Results: The SNF2L gene spans 77 kb and is encoded by 25 exons that undergo alternate splicing to generate several distinct transcripts. Specific isoforms are generated through the alternate use of exons 1 and 13, and by the use of alternate donor splice sites within exon 24. Alternate splicing within exon 24 removes a NLS sequence and alters the subcellular distribution of the SNF2L protein. We identified 3 single nucleotide polymorphisms but no mutations in our 12 patients.

Conclusion: Our results demonstrate that there are numerous splice variants of SNF2L that are expressed in multiple cell types and which alter subcellular localization and function. SNF2L mutations are not a cause of XLMR in our cohort of patients, although we cannot exclude the possibility that regulatory mutations might exist. Nonetheless, SNF2L remains a candidate for XLMR localized to Xq25-26, including the Shashi XLMR syndrome.

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Alternative splicing of exons 24 and 25 generates nuclear and cytoplasmic isoforms of SNF2L. Alternative splicing of the SNF2L gene at the 3' end generates a transcript containing either the full sequence of exons 24 and 25, which encodes a shorter form of SNF2L without a nuclear localization signal (SNF2LΔNLS), or a transcript lacking 100 bp that encodes for a larger protein isoform (SNF2L) with an NLS (underlined). B. RT-PCR analysis demonstrated that both 3' variants are present in most cells and tissues examined, while the NLS isoform is predominant. M, marker; -D, no DNA template; -RT, no reverse transcriptase. Lanes 1–5, human cell lines and fetal brain sample as follows: 1, 293-HEK cells; 2, SH-SY5Y cells; 3, NT2 cells; 4, hNT neurons; 5, human fetal brain. Lanes 6–13, human brain regions including: 6, amygdala; 7, basal ganglia; 8, caudate nucleus; 9, cerebellum; 10, frontal cortex; 11, hippocampus; 12, pons; 13, thalamus. Lanes 14–20, human tissue samples including: 14, heart; 15, kidney; 16, liver; 17, ovary; 18, placenta; 19, skeletal muscle; and 20, testes. C. Indirect immunofluorescence imaging of 293 HEK cells transfected with FLAG-epitope tagged SNF2LΔNLS and SNF2L were stained with anti-flag antibody (green) or DAPI (blue). Note that SNF2LΔNLS encodes a protein that is localized exclusively in the cytoplasm while SNF2L is expressed only in the nucleus (arrows point to nuclei of transfected cells).
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Figure 2: Alternative splicing of exons 24 and 25 generates nuclear and cytoplasmic isoforms of SNF2L. Alternative splicing of the SNF2L gene at the 3' end generates a transcript containing either the full sequence of exons 24 and 25, which encodes a shorter form of SNF2L without a nuclear localization signal (SNF2LΔNLS), or a transcript lacking 100 bp that encodes for a larger protein isoform (SNF2L) with an NLS (underlined). B. RT-PCR analysis demonstrated that both 3' variants are present in most cells and tissues examined, while the NLS isoform is predominant. M, marker; -D, no DNA template; -RT, no reverse transcriptase. Lanes 1–5, human cell lines and fetal brain sample as follows: 1, 293-HEK cells; 2, SH-SY5Y cells; 3, NT2 cells; 4, hNT neurons; 5, human fetal brain. Lanes 6–13, human brain regions including: 6, amygdala; 7, basal ganglia; 8, caudate nucleus; 9, cerebellum; 10, frontal cortex; 11, hippocampus; 12, pons; 13, thalamus. Lanes 14–20, human tissue samples including: 14, heart; 15, kidney; 16, liver; 17, ovary; 18, placenta; 19, skeletal muscle; and 20, testes. C. Indirect immunofluorescence imaging of 293 HEK cells transfected with FLAG-epitope tagged SNF2LΔNLS and SNF2L were stained with anti-flag antibody (green) or DAPI (blue). Note that SNF2LΔNLS encodes a protein that is localized exclusively in the cytoplasm while SNF2L is expressed only in the nucleus (arrows point to nuclei of transfected cells).

Mentions: In addition to the variation at the amino-terminus, the human and mouse amino acid sequences also differed at their carboxyl-terminus following Blast tool analysis. The last seven amino acids of the human sequence were not present in the mouse SNF2L protein sequence, but instead were replaced by 23 unique residues (Figure 2A). RT-PCR analysis demonstrated that both 3'-end variants could be detected in a variety of human cell lines, tissues and specific brain regions with the NLS-containing isoform representing the predominant species (Figure 2B). Sequencing demonstrated that the two transcripts are generated by alternative splicing of exons 24 and 25. Inclusion of exons 24 and 25 result in a shorter peptide due to a stop codon located at the start of exon 25. We called this variant SNF2LΔNLS. Alternative use of splice sites within exons 24 and 25 removes 100 bp and generates a transcript that contains an additional 23 amino acids similar to the reported mouse protein (and herein called SNF2L). More importantly, we used the PredictNLS program [26] to determine that this alternatively spliced region contained a putative NLS sequence. To verify the importance of the NLS in vivo, we transiently transfected 293 HEK cells with FLAG-epitope tagged SNF2L or SNF2LΔNLS and detected the expressed protein by indirect immunofluorescence. As shown in Figure 2C, SNF2LΔNLS localized exclusively in the cytoplasm whereas SNF2L was present in the nucleus.


Characterization of novel isoforms and evaluation of SNF2L/SMARCA1 as a candidate gene for X-linked mental retardation in 12 families linked to Xq25-26.

Lazzaro MA, Todd MA, Lavigne P, Vallee D, De Maria A, Picketts DJ - BMC Med. Genet. (2008)

Alternative splicing of exons 24 and 25 generates nuclear and cytoplasmic isoforms of SNF2L. Alternative splicing of the SNF2L gene at the 3' end generates a transcript containing either the full sequence of exons 24 and 25, which encodes a shorter form of SNF2L without a nuclear localization signal (SNF2LΔNLS), or a transcript lacking 100 bp that encodes for a larger protein isoform (SNF2L) with an NLS (underlined). B. RT-PCR analysis demonstrated that both 3' variants are present in most cells and tissues examined, while the NLS isoform is predominant. M, marker; -D, no DNA template; -RT, no reverse transcriptase. Lanes 1–5, human cell lines and fetal brain sample as follows: 1, 293-HEK cells; 2, SH-SY5Y cells; 3, NT2 cells; 4, hNT neurons; 5, human fetal brain. Lanes 6–13, human brain regions including: 6, amygdala; 7, basal ganglia; 8, caudate nucleus; 9, cerebellum; 10, frontal cortex; 11, hippocampus; 12, pons; 13, thalamus. Lanes 14–20, human tissue samples including: 14, heart; 15, kidney; 16, liver; 17, ovary; 18, placenta; 19, skeletal muscle; and 20, testes. C. Indirect immunofluorescence imaging of 293 HEK cells transfected with FLAG-epitope tagged SNF2LΔNLS and SNF2L were stained with anti-flag antibody (green) or DAPI (blue). Note that SNF2LΔNLS encodes a protein that is localized exclusively in the cytoplasm while SNF2L is expressed only in the nucleus (arrows point to nuclei of transfected cells).
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Figure 2: Alternative splicing of exons 24 and 25 generates nuclear and cytoplasmic isoforms of SNF2L. Alternative splicing of the SNF2L gene at the 3' end generates a transcript containing either the full sequence of exons 24 and 25, which encodes a shorter form of SNF2L without a nuclear localization signal (SNF2LΔNLS), or a transcript lacking 100 bp that encodes for a larger protein isoform (SNF2L) with an NLS (underlined). B. RT-PCR analysis demonstrated that both 3' variants are present in most cells and tissues examined, while the NLS isoform is predominant. M, marker; -D, no DNA template; -RT, no reverse transcriptase. Lanes 1–5, human cell lines and fetal brain sample as follows: 1, 293-HEK cells; 2, SH-SY5Y cells; 3, NT2 cells; 4, hNT neurons; 5, human fetal brain. Lanes 6–13, human brain regions including: 6, amygdala; 7, basal ganglia; 8, caudate nucleus; 9, cerebellum; 10, frontal cortex; 11, hippocampus; 12, pons; 13, thalamus. Lanes 14–20, human tissue samples including: 14, heart; 15, kidney; 16, liver; 17, ovary; 18, placenta; 19, skeletal muscle; and 20, testes. C. Indirect immunofluorescence imaging of 293 HEK cells transfected with FLAG-epitope tagged SNF2LΔNLS and SNF2L were stained with anti-flag antibody (green) or DAPI (blue). Note that SNF2LΔNLS encodes a protein that is localized exclusively in the cytoplasm while SNF2L is expressed only in the nucleus (arrows point to nuclei of transfected cells).
Mentions: In addition to the variation at the amino-terminus, the human and mouse amino acid sequences also differed at their carboxyl-terminus following Blast tool analysis. The last seven amino acids of the human sequence were not present in the mouse SNF2L protein sequence, but instead were replaced by 23 unique residues (Figure 2A). RT-PCR analysis demonstrated that both 3'-end variants could be detected in a variety of human cell lines, tissues and specific brain regions with the NLS-containing isoform representing the predominant species (Figure 2B). Sequencing demonstrated that the two transcripts are generated by alternative splicing of exons 24 and 25. Inclusion of exons 24 and 25 result in a shorter peptide due to a stop codon located at the start of exon 25. We called this variant SNF2LΔNLS. Alternative use of splice sites within exons 24 and 25 removes 100 bp and generates a transcript that contains an additional 23 amino acids similar to the reported mouse protein (and herein called SNF2L). More importantly, we used the PredictNLS program [26] to determine that this alternatively spliced region contained a putative NLS sequence. To verify the importance of the NLS in vivo, we transiently transfected 293 HEK cells with FLAG-epitope tagged SNF2L or SNF2LΔNLS and detected the expressed protein by indirect immunofluorescence. As shown in Figure 2C, SNF2LΔNLS localized exclusively in the cytoplasm whereas SNF2L was present in the nucleus.

Bottom Line: Our results demonstrate that there are numerous splice variants of SNF2L that are expressed in multiple cell types and which alter subcellular localization and function.SNF2L mutations are not a cause of XLMR in our cohort of patients, although we cannot exclude the possibility that regulatory mutations might exist.Nonetheless, SNF2L remains a candidate for XLMR localized to Xq25-26, including the Shashi XLMR syndrome.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ottawa Health Research Institute, 501 Smyth Road, Ottawa, ON K1H 8L6, Canada. maribeth_lazzaro@hc-sc.gc.ca

ABSTRACT

Background: Mutations in genes whose products modify chromatin structure have been recognized as a cause of X-linked mental retardation (XLMR). These genes encode proteins that regulate DNA methylation (MeCP2), modify histones (RSK2 and JARID1C), and remodel nucleosomes through ATP hydrolysis (ATRX). Thus, genes encoding other chromatin modifying proteins should also be considered as disease candidate genes. In this work, we have characterized the SNF2L gene, encoding an ATP-dependent chromatin remodeling protein of the ISWI family, and sequenced the gene in patients from 12 XLMR families linked to Xq25-26.

Methods: We used an in silico and RT-PCR approach to fully characterize specific SNF2L isoforms. Mutation screening was performed in 12 patients from individual families with syndromic or non-syndromic XLMR. We sequenced each of the 25 exons encompassing the entire coding region, complete 5' and 3' untranslated regions, and consensus splice-sites.

Results: The SNF2L gene spans 77 kb and is encoded by 25 exons that undergo alternate splicing to generate several distinct transcripts. Specific isoforms are generated through the alternate use of exons 1 and 13, and by the use of alternate donor splice sites within exon 24. Alternate splicing within exon 24 removes a NLS sequence and alters the subcellular distribution of the SNF2L protein. We identified 3 single nucleotide polymorphisms but no mutations in our 12 patients.

Conclusion: Our results demonstrate that there are numerous splice variants of SNF2L that are expressed in multiple cell types and which alter subcellular localization and function. SNF2L mutations are not a cause of XLMR in our cohort of patients, although we cannot exclude the possibility that regulatory mutations might exist. Nonetheless, SNF2L remains a candidate for XLMR localized to Xq25-26, including the Shashi XLMR syndrome.

Show MeSH
Related in: MedlinePlus