Accepted Manuscript Identification of Nuclear Localization Signals within the human BCOR protein Thunyaporn Surapornsawasd, Takuya Ogawa, Keiji Moriyama PII: DOI: Reference:
S0014-5793(15)00447-0 http://dx.doi.org/10.1016/j.febslet.2015.05.046 FEBS 37202
To appear in:
FEBS Letters
Received Date: Accepted Date:
25 March 2015 12 May 2015
Please cite this article as: Surapornsawasd, T., Ogawa, T., Moriyama, K., Identification of Nuclear Localization Signals within the human BCOR protein, FEBS Letters (2015), doi: http://dx.doi.org/10.1016/j.febslet.2015.05.046
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Identification of Nuclear Localization Signals within the human BCOR Protein
Thunyaporn Surapornsawasd1, Takuya Ogawa1,2, Keiji Moriyama1,2 1
Maxillofacial Orthognathics, Graduate School, Tokyo Medical and Dental University,
1-5-45 Yushima, Bunkyo-ku, Tokyo, Japan, 113-8549 2
Hard Tissue Genome Research Center, Tokyo Medical and Dental University, Tokyo,
1-5-45 Yushima, Bunkyo-ku, Tokyo, Japan, 113-8549 E-mail address: Thunyaporn Surapornsawasd : [email protected] Takuya Ogawa: [email protected] Keiji Moriyamal: [email protected]
Address correspondence to: Takuya Ogawa, Maxillofacial Orthognathics, Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, Japan 113-8549. E-mail [email protected]. Tel and Fax: 81-3-5803-5533
Identification of Nuclear Localization Signals within the human BCOR protein
Abstract Mutations in the BCL-6 corepressor (BCOR) gene, which encodes a transcriptional corepressor, were described to cause oculofaciocardiodental syndrome (MIM 300166). The purpose of this study was to localize the classical nuclear localization signals (NLSs) of the BCOR using reported human BCOR mutations with comparable phenotypes. The genotype-phenotype correlation among the mutations could not be clearly explained; however, the classical NLSs were identified at two possible sites; RVDRKRKVSGD at aa1131–1141 (NLS1) and LKAKRRRVSK at aa1158–1167 (NLS2). In addition, according to our results, NLS2 displayed a more efficient nuclear import function than NLS1.
Keywords: BCOR, Oculofaciocardiofacial syndrome, X-linked dominant hereditary, Nuclear localization, Mutagenesis
Research Highlights
We generated the seven reported BCOR mutations with comparable phenotypes.
We identified two classical NLSs of the BCOR.
BCOR classical NLSs were located at aa1131–1141 (NLS1) and aa1158–1167 (NLS2).
NLS2 displayed a more efficient nuclear import function than NLS1.
1. Introduction Oculofaciocardiodental syndrome (OFCD; MIM 300166) is a rare disorder characterized by microphthalmia, congenital cataracts, facial dysmorphic features, congenital heart defects, and dental anomalies [1-6]. This syndrome is an X-linked dominant, male-specific lethal with skewed X inactivation in heterozygous female individuals [1,7]. Mutations in the BCL-6 corepressor (BCOR) gene were described to cause OFCD syndrome [3]. Many mutations in the BCOR gene have been reported in OFCD patients. The reported mutations were dispersed along the entire 15 exons of the BCOR gene and most of these mutations were predicted to generate premature stop codons. Regardless of the mutation site, all patients have similar typical OFCD phenotypes, but differ in severity even in single families with the same mutation [8]. The BCOR gene was first identified as a corepressor that interacts specifically with the POZ domain of BCL6 [9]. The BCOR gene is expressed as several alternatively spliced transcripts [10]. To date, the BCOR gene is known to encode at least four important isoforms: isoform a (NP_001116855.1), isoform b (NP_001116856.1), isoform c (NP_001116857.1), and isoform d (Genbank: AAG41430.1). Because BCOR protein is a transcriptional corepressor, it needs to be imported into the nucleus to execute its proper activity. In eukaryotic cells, the genetic material and transcriptional machinery of the nucleus are separated from the translational machinery and metabolic systems of the cytoplasm by the nuclear envelope. Transport of macromolecules into and out of the nucleus occurs through large proteinaceous structures called nuclear pore complexes. Ions and small proteins (A
7 8 9 10
11
12 13 14
15
BCOR gene Mut3: c.2926C>T
Mut5: c.3427_3428insA
Mut4: c.3286delG
b Mut1: p.W191Gfs*25
Mut2: p.F871Lfs*8
Mut6: p.S1223Wfs*15 NSPC1
Bcl6
Normal BCOR protein
AF9
Ankyrin 498
Mut3: p.R976*
1157
514
1251
Mut4: p.E1096Rfs*16
Mut5: p.S1143Kfs*4
Bcl6: region that interact with Bcl6 AF9: region that interact with AF9, the common mixed lineage leukemia gene fusion partner NSPC1: region that interact with nervous system polycomb1 Ankyrin: Ankyrin repeats that involved in protein-protein interaction, protein structure
c
WT Mut1 Mut2 Mut3 Mut4 Mut5 Mut6 Mut7
188.23 kDa 22.91 kDa 93.93 kDa 104.12 kDa 119.92 kDa 123.60 kDa 134.18 kDa 174.25 kDa Empty plasmid
WT
Mut2
Mut1
Mut4
Mut3
Mut5
Mut6
Mut7
BCOR 188.23 kDa 174.25 kDa 134.18 kDa 123.60 kDa 119.92 kDa 104.12 kDa 93.93 kDa
22.91 kDa
β-actin 43 kDa
*
*
*
*
*
*
*
*
Mut7: p.W1598*
Fig.2 Subcellular localization of the mutant proteins DAPI
FITC
Merge
Fluorescence intensity 1
Empty plasmid
0.8 0.6 0.4 0.2 0 Nucleus
Cytoplasm
** 30 25
WT
20 15 10 5 0 Nucleus
Cytoplasm
Nucleus
Cytoplasm
50 40
Mut1
30 20 10 0
** 30 25 20
Mut2
15 10 5 0 Nucleus
Cytoplasm
** 40 30
Mut3
20 10 0 Nucleus
Cytoplasm
** 50 40
Mut4
30 20 10 0 Nucleus
Cytoplasm
40 30
Mut5
20 10 0 Nucleus
Cytoplasm
** 40 30
Mut6
20 10 0 Nucleus
Cytoplasm
** 30 25
Mut7
20 15 10 5 0 Nucleus
Cytoplasm
Fig.3 The possible NLS sites
a Mut5
Orthologues of BCOR protein at aa1125-1175 1125
Homo sapiens: Pan troglodytes: Gorilla gorilla gorilla: Nomascus leucogenys: Callithrix jacchus: Pongo abelii: Macaca mulatta: Canis lupus familiaris: Felis catus: Mus musculus: Rattus norvegicus:
NLS2
NLS1
1175
PHSPTLRVDRKRKVSGDSSHTETTAEEVPEDPLLKAKRRRVSKGLHPKKQR ! PHSPTLRVDRKRKVSGDSSHTETTAEEVPEDPLLKAKRRRVSKGLHPKKQR ! PHSPTLRVDRKRKVSGDSSHTETTAEEVPEDPLLKAKRRRVSKGLHPKKQR ! LHSPTLRVDRKRKVSGDSSHTETTAEEVPEDPLLKAKRRRVSKGLHPKKQR ! PPSPTLRVDRKRKVSGDSSHTETTAEEVPEDPLLKAKRRRVSKGLHPKKQR! PHSPTLRVDRKRKVSGDSSHTETTAEEVPEDPLLKAKRRRVFKGLHPKKQR! PRSPTLRVDRKRKVSGDSSHTETTAEEVPEDPLLKAKRRRVSKGLHPKKQR ! PHSPTLRLDRKRKVSGDSTHTETAAEELPEDPLLKAKRRRVSKGLHPKKQR ! PHSPTLRLDRKRKVSGDSNHTETAAEDLPEDPLLKAKRRRVSKGLHPKKQR ! PHSPTLRLDRKRKLSGDSTHTETAVEELAEDP-LKAKRRRISKGLHPKKQR ! SHSPTLRLDRKRKASGDSTHTETTVEELTEDP-LKAKRRRISKGLHPKKQR!
b Mut1
Mut2 Mut3 Mut4
Mut6
Mut5
NLS1
Mut7
NLS2
Normal BCOR protein
1131 C=N C>N C=N C
S0014-5793(15)00447-0 http://dx.doi.org/10.1016/j.febslet.2015.05.046 FEBS 37202
To appear in:
FEBS Letters
Received Date: Accepted Date:
25 March 2015 12 May 2015
Please cite this article as: Surapornsawasd, T., Ogawa, T., Moriyama, K., Identification of Nuclear Localization Signals within the human BCOR protein, FEBS Letters (2015), doi: http://dx.doi.org/10.1016/j.febslet.2015.05.046
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Identification of Nuclear Localization Signals within the human BCOR Protein
Thunyaporn Surapornsawasd1, Takuya Ogawa1,2, Keiji Moriyama1,2 1
Maxillofacial Orthognathics, Graduate School, Tokyo Medical and Dental University,
1-5-45 Yushima, Bunkyo-ku, Tokyo, Japan, 113-8549 2
Hard Tissue Genome Research Center, Tokyo Medical and Dental University, Tokyo,
1-5-45 Yushima, Bunkyo-ku, Tokyo, Japan, 113-8549 E-mail address: Thunyaporn Surapornsawasd : [email protected] Takuya Ogawa: [email protected] Keiji Moriyamal: [email protected]
Address correspondence to: Takuya Ogawa, Maxillofacial Orthognathics, Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, Japan 113-8549. E-mail [email protected]. Tel and Fax: 81-3-5803-5533
Identification of Nuclear Localization Signals within the human BCOR protein
Abstract Mutations in the BCL-6 corepressor (BCOR) gene, which encodes a transcriptional corepressor, were described to cause oculofaciocardiodental syndrome (MIM 300166). The purpose of this study was to localize the classical nuclear localization signals (NLSs) of the BCOR using reported human BCOR mutations with comparable phenotypes. The genotype-phenotype correlation among the mutations could not be clearly explained; however, the classical NLSs were identified at two possible sites; RVDRKRKVSGD at aa1131–1141 (NLS1) and LKAKRRRVSK at aa1158–1167 (NLS2). In addition, according to our results, NLS2 displayed a more efficient nuclear import function than NLS1.
Keywords: BCOR, Oculofaciocardiofacial syndrome, X-linked dominant hereditary, Nuclear localization, Mutagenesis
Research Highlights
We generated the seven reported BCOR mutations with comparable phenotypes.
We identified two classical NLSs of the BCOR.
BCOR classical NLSs were located at aa1131–1141 (NLS1) and aa1158–1167 (NLS2).
NLS2 displayed a more efficient nuclear import function than NLS1.
1. Introduction Oculofaciocardiodental syndrome (OFCD; MIM 300166) is a rare disorder characterized by microphthalmia, congenital cataracts, facial dysmorphic features, congenital heart defects, and dental anomalies [1-6]. This syndrome is an X-linked dominant, male-specific lethal with skewed X inactivation in heterozygous female individuals [1,7]. Mutations in the BCL-6 corepressor (BCOR) gene were described to cause OFCD syndrome [3]. Many mutations in the BCOR gene have been reported in OFCD patients. The reported mutations were dispersed along the entire 15 exons of the BCOR gene and most of these mutations were predicted to generate premature stop codons. Regardless of the mutation site, all patients have similar typical OFCD phenotypes, but differ in severity even in single families with the same mutation [8]. The BCOR gene was first identified as a corepressor that interacts specifically with the POZ domain of BCL6 [9]. The BCOR gene is expressed as several alternatively spliced transcripts [10]. To date, the BCOR gene is known to encode at least four important isoforms: isoform a (NP_001116855.1), isoform b (NP_001116856.1), isoform c (NP_001116857.1), and isoform d (Genbank: AAG41430.1). Because BCOR protein is a transcriptional corepressor, it needs to be imported into the nucleus to execute its proper activity. In eukaryotic cells, the genetic material and transcriptional machinery of the nucleus are separated from the translational machinery and metabolic systems of the cytoplasm by the nuclear envelope. Transport of macromolecules into and out of the nucleus occurs through large proteinaceous structures called nuclear pore complexes. Ions and small proteins (A
7 8 9 10
11
12 13 14
15
BCOR gene Mut3: c.2926C>T
Mut5: c.3427_3428insA
Mut4: c.3286delG
b Mut1: p.W191Gfs*25
Mut2: p.F871Lfs*8
Mut6: p.S1223Wfs*15 NSPC1
Bcl6
Normal BCOR protein
AF9
Ankyrin 498
Mut3: p.R976*
1157
514
1251
Mut4: p.E1096Rfs*16
Mut5: p.S1143Kfs*4
Bcl6: region that interact with Bcl6 AF9: region that interact with AF9, the common mixed lineage leukemia gene fusion partner NSPC1: region that interact with nervous system polycomb1 Ankyrin: Ankyrin repeats that involved in protein-protein interaction, protein structure
c
WT Mut1 Mut2 Mut3 Mut4 Mut5 Mut6 Mut7
188.23 kDa 22.91 kDa 93.93 kDa 104.12 kDa 119.92 kDa 123.60 kDa 134.18 kDa 174.25 kDa Empty plasmid
WT
Mut2
Mut1
Mut4
Mut3
Mut5
Mut6
Mut7
BCOR 188.23 kDa 174.25 kDa 134.18 kDa 123.60 kDa 119.92 kDa 104.12 kDa 93.93 kDa
22.91 kDa
β-actin 43 kDa
*
*
*
*
*
*
*
*
Mut7: p.W1598*
Fig.2 Subcellular localization of the mutant proteins DAPI
FITC
Merge
Fluorescence intensity 1
Empty plasmid
0.8 0.6 0.4 0.2 0 Nucleus
Cytoplasm
** 30 25
WT
20 15 10 5 0 Nucleus
Cytoplasm
Nucleus
Cytoplasm
50 40
Mut1
30 20 10 0
** 30 25 20
Mut2
15 10 5 0 Nucleus
Cytoplasm
** 40 30
Mut3
20 10 0 Nucleus
Cytoplasm
** 50 40
Mut4
30 20 10 0 Nucleus
Cytoplasm
40 30
Mut5
20 10 0 Nucleus
Cytoplasm
** 40 30
Mut6
20 10 0 Nucleus
Cytoplasm
** 30 25
Mut7
20 15 10 5 0 Nucleus
Cytoplasm
Fig.3 The possible NLS sites
a Mut5
Orthologues of BCOR protein at aa1125-1175 1125
Homo sapiens: Pan troglodytes: Gorilla gorilla gorilla: Nomascus leucogenys: Callithrix jacchus: Pongo abelii: Macaca mulatta: Canis lupus familiaris: Felis catus: Mus musculus: Rattus norvegicus:
NLS2
NLS1
1175
PHSPTLRVDRKRKVSGDSSHTETTAEEVPEDPLLKAKRRRVSKGLHPKKQR ! PHSPTLRVDRKRKVSGDSSHTETTAEEVPEDPLLKAKRRRVSKGLHPKKQR ! PHSPTLRVDRKRKVSGDSSHTETTAEEVPEDPLLKAKRRRVSKGLHPKKQR ! LHSPTLRVDRKRKVSGDSSHTETTAEEVPEDPLLKAKRRRVSKGLHPKKQR ! PPSPTLRVDRKRKVSGDSSHTETTAEEVPEDPLLKAKRRRVSKGLHPKKQR! PHSPTLRVDRKRKVSGDSSHTETTAEEVPEDPLLKAKRRRVFKGLHPKKQR! PRSPTLRVDRKRKVSGDSSHTETTAEEVPEDPLLKAKRRRVSKGLHPKKQR ! PHSPTLRLDRKRKVSGDSTHTETAAEELPEDPLLKAKRRRVSKGLHPKKQR ! PHSPTLRLDRKRKVSGDSNHTETAAEDLPEDPLLKAKRRRVSKGLHPKKQR ! PHSPTLRLDRKRKLSGDSTHTETAVEELAEDP-LKAKRRRISKGLHPKKQR ! SHSPTLRLDRKRKASGDSTHTETTVEELTEDP-LKAKRRRISKGLHPKKQR!
b Mut1
Mut2 Mut3 Mut4
Mut6
Mut5
NLS1
Mut7
NLS2
Normal BCOR protein
1131 C=N C>N C=N C
