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Drosophila muscleblind is involved in troponin T alternative splicing and apoptosis.

Vicente-Crespo M, Pascual M, Fernandez-Costa JM, Garcia-Lopez A, Monferrer L, Miranda ME, Zhou L, Artero RD - PLoS ONE (2008)

Bottom Line: We found missplicing of troponin T in muscleblind mutant pupae and confirmed Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3) minigene splicing in human HEK cells.MblC overexpression in the wing imaginal disc activated apoptosis in a spatially restricted manner.Site-directed mutagenesis of the motif revealed no change in activity of mutant MblC on TnnT3 minigene splicing or aberrant binding to CUG repeat RNA, but altered the ability of the protein to form perinuclear aggregates and enhanced cell death-inducing activity of MblC overexpression.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Valencia, Valencia, Spain.

ABSTRACT

Background: Muscleblind-like proteins (MBNL) have been involved in a developmental switch in the use of defined cassette exons. Such transition fails in the CTG repeat expansion disease myotonic dystrophy due, in part, to sequestration of MBNL proteins by CUG repeat RNA. Four protein isoforms (MblA-D) are coded by the unique Drosophila muscleblind gene.

Methodology/principal findings: We used evolutionary, genetic and cell culture approaches to study muscleblind (mbl) function in flies. The evolutionary study showed that the MblC protein isoform was readily conserved from nematods to Drosophila, which suggests that it performs the most ancestral muscleblind functions. Overexpression of MblC in the fly eye precursors led to an externally rough eye morphology. This phenotype was used in a genetic screen to identify five dominant suppressors and 13 dominant enhancers including Drosophila CUG-BP1 homolog aret, exon junction complex components tsunagi and Aly, and pro-apoptotic genes Traf1 and reaper. We further investigated Muscleblind implication in apoptosis and splicing regulation. We found missplicing of troponin T in muscleblind mutant pupae and confirmed Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3) minigene splicing in human HEK cells. MblC overexpression in the wing imaginal disc activated apoptosis in a spatially restricted manner. Bioinformatics analysis identified a conserved FKRP motif, weakly resembling a sumoylation target site, in the MblC-specific sequence. Site-directed mutagenesis of the motif revealed no change in activity of mutant MblC on TnnT3 minigene splicing or aberrant binding to CUG repeat RNA, but altered the ability of the protein to form perinuclear aggregates and enhanced cell death-inducing activity of MblC overexpression.

Conclusions/significance: Taken together our genetic approach identify cellular processes influenced by Muscleblind function, whereas in vivo and cell culture experiments define Drosophila troponin T as a new Muscleblind target, reveal a potential involvement of MblC in programmed cell death and recognize the FKRP motif as a putative regulator of MblC function and/or subcellular location in the cell.

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Mutation of a conserved FKRP motif reduces nuclear localization and enhances cell death-inducing activity of MblC.(A) ClustalW multiple alignment of part of Drosophila MblC-specific sequence (dmel) with homologous sequences from C. elegans (cel), Anopheles (aga), Danio rerio (dre) mus musculus (mmu; Mbnl1) and humans (hsa; MBNL1). SUMOplot web server predicts the Drosophila FKRP and C. elegans MKRP sequences (boxed) as sumoylation target sites. A conserved lysine (asterisk) was mutated to isoleucine in MblCK202I. (B) COSM6 cells transfected with 1 µg of GFP-tagged MblC protein showed preferential nuclear localization and perinuclear aggregates that increased in number in MblCK202I. (B) HEK293T cells transfected with 300 ng of GFP-tagged MblC showed no perinuclear aggregates whereas MblCΚ202Ι still aggregated. Mutant MblC (green) co-localized with CUG ribonuclear foci (red) in the cell nucleus (blue) stained with DAPI (C). (D) TnnT3 minigene splicing assay in HEK293T cells. GFP-tagged MblC and MblCK202I promoted foetal exon exclusion to the same extent compared to transfection of the empty vector (p<0.01). (E) Drosophila S2 cell viability assay 48 h after transfection of normal and mutant MblC. MblCK202I significantly reduced the number of viable cells (y-axis) compared to transfection of vector alone (p<0.05).
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pone-0001613-g004: Mutation of a conserved FKRP motif reduces nuclear localization and enhances cell death-inducing activity of MblC.(A) ClustalW multiple alignment of part of Drosophila MblC-specific sequence (dmel) with homologous sequences from C. elegans (cel), Anopheles (aga), Danio rerio (dre) mus musculus (mmu; Mbnl1) and humans (hsa; MBNL1). SUMOplot web server predicts the Drosophila FKRP and C. elegans MKRP sequences (boxed) as sumoylation target sites. A conserved lysine (asterisk) was mutated to isoleucine in MblCK202I. (B) COSM6 cells transfected with 1 µg of GFP-tagged MblC protein showed preferential nuclear localization and perinuclear aggregates that increased in number in MblCK202I. (B) HEK293T cells transfected with 300 ng of GFP-tagged MblC showed no perinuclear aggregates whereas MblCΚ202Ι still aggregated. Mutant MblC (green) co-localized with CUG ribonuclear foci (red) in the cell nucleus (blue) stained with DAPI (C). (D) TnnT3 minigene splicing assay in HEK293T cells. GFP-tagged MblC and MblCK202I promoted foetal exon exclusion to the same extent compared to transfection of the empty vector (p<0.01). (E) Drosophila S2 cell viability assay 48 h after transfection of normal and mutant MblC. MblCK202I significantly reduced the number of viable cells (y-axis) compared to transfection of vector alone (p<0.05).

Mentions: Drosophila Muscleblind protein isoforms sharing both their zinc fingers (MblA-C) showed different behaviour in various functional assays including differences in subcellular localization and splicing activity when over-expressed in vertebrate COSM6 cells [27]. Bioinformatics analysis of the MblC-specific sequence (64 amino acids) identified a region of conservation in distantly related Muscleblind proteins, ranging from C. elegans to vertebrate and human homologs (Figure 4A and Figure S1). SUMOplot, a sumoylation site prediction web server [44], identified the FKRP in Drosophila and MKRP in C. elegans as putative sumoylation sites. Small ubiquitin-related modifier (Sumo) is a 10 kDa post-translational modification that typically does not lead to protein degradation but changes in intracellular localization of proteins [45], [46]. Western blotting of protein extracts from S2 cells transfected with myc-tagged Muscleblind proteins, however, did not reveal bands of higher than predicted molecular weight (Figure 3G). Therefore, if sumoylation is actually taking place, must affect a very small proportion of the MblC protein isoform. As an alternative approach, and in order to test the relevance of the FKRP site in MblC function, we mutated lysine 201 into isoleucine by site-directed mutagenesis (Figure 4A) and tested the mutant protein (MblCΚ202Ι) in the functional assays we performed before in vertebrate and Drosophila cells with wild type MblC.


Drosophila muscleblind is involved in troponin T alternative splicing and apoptosis.

Vicente-Crespo M, Pascual M, Fernandez-Costa JM, Garcia-Lopez A, Monferrer L, Miranda ME, Zhou L, Artero RD - PLoS ONE (2008)

Mutation of a conserved FKRP motif reduces nuclear localization and enhances cell death-inducing activity of MblC.(A) ClustalW multiple alignment of part of Drosophila MblC-specific sequence (dmel) with homologous sequences from C. elegans (cel), Anopheles (aga), Danio rerio (dre) mus musculus (mmu; Mbnl1) and humans (hsa; MBNL1). SUMOplot web server predicts the Drosophila FKRP and C. elegans MKRP sequences (boxed) as sumoylation target sites. A conserved lysine (asterisk) was mutated to isoleucine in MblCK202I. (B) COSM6 cells transfected with 1 µg of GFP-tagged MblC protein showed preferential nuclear localization and perinuclear aggregates that increased in number in MblCK202I. (B) HEK293T cells transfected with 300 ng of GFP-tagged MblC showed no perinuclear aggregates whereas MblCΚ202Ι still aggregated. Mutant MblC (green) co-localized with CUG ribonuclear foci (red) in the cell nucleus (blue) stained with DAPI (C). (D) TnnT3 minigene splicing assay in HEK293T cells. GFP-tagged MblC and MblCK202I promoted foetal exon exclusion to the same extent compared to transfection of the empty vector (p<0.01). (E) Drosophila S2 cell viability assay 48 h after transfection of normal and mutant MblC. MblCK202I significantly reduced the number of viable cells (y-axis) compared to transfection of vector alone (p<0.05).
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getmorefigures.php?uid=PMC2238819&req=5

pone-0001613-g004: Mutation of a conserved FKRP motif reduces nuclear localization and enhances cell death-inducing activity of MblC.(A) ClustalW multiple alignment of part of Drosophila MblC-specific sequence (dmel) with homologous sequences from C. elegans (cel), Anopheles (aga), Danio rerio (dre) mus musculus (mmu; Mbnl1) and humans (hsa; MBNL1). SUMOplot web server predicts the Drosophila FKRP and C. elegans MKRP sequences (boxed) as sumoylation target sites. A conserved lysine (asterisk) was mutated to isoleucine in MblCK202I. (B) COSM6 cells transfected with 1 µg of GFP-tagged MblC protein showed preferential nuclear localization and perinuclear aggregates that increased in number in MblCK202I. (B) HEK293T cells transfected with 300 ng of GFP-tagged MblC showed no perinuclear aggregates whereas MblCΚ202Ι still aggregated. Mutant MblC (green) co-localized with CUG ribonuclear foci (red) in the cell nucleus (blue) stained with DAPI (C). (D) TnnT3 minigene splicing assay in HEK293T cells. GFP-tagged MblC and MblCK202I promoted foetal exon exclusion to the same extent compared to transfection of the empty vector (p<0.01). (E) Drosophila S2 cell viability assay 48 h after transfection of normal and mutant MblC. MblCK202I significantly reduced the number of viable cells (y-axis) compared to transfection of vector alone (p<0.05).
Mentions: Drosophila Muscleblind protein isoforms sharing both their zinc fingers (MblA-C) showed different behaviour in various functional assays including differences in subcellular localization and splicing activity when over-expressed in vertebrate COSM6 cells [27]. Bioinformatics analysis of the MblC-specific sequence (64 amino acids) identified a region of conservation in distantly related Muscleblind proteins, ranging from C. elegans to vertebrate and human homologs (Figure 4A and Figure S1). SUMOplot, a sumoylation site prediction web server [44], identified the FKRP in Drosophila and MKRP in C. elegans as putative sumoylation sites. Small ubiquitin-related modifier (Sumo) is a 10 kDa post-translational modification that typically does not lead to protein degradation but changes in intracellular localization of proteins [45], [46]. Western blotting of protein extracts from S2 cells transfected with myc-tagged Muscleblind proteins, however, did not reveal bands of higher than predicted molecular weight (Figure 3G). Therefore, if sumoylation is actually taking place, must affect a very small proportion of the MblC protein isoform. As an alternative approach, and in order to test the relevance of the FKRP site in MblC function, we mutated lysine 201 into isoleucine by site-directed mutagenesis (Figure 4A) and tested the mutant protein (MblCΚ202Ι) in the functional assays we performed before in vertebrate and Drosophila cells with wild type MblC.

Bottom Line: We found missplicing of troponin T in muscleblind mutant pupae and confirmed Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3) minigene splicing in human HEK cells.MblC overexpression in the wing imaginal disc activated apoptosis in a spatially restricted manner.Site-directed mutagenesis of the motif revealed no change in activity of mutant MblC on TnnT3 minigene splicing or aberrant binding to CUG repeat RNA, but altered the ability of the protein to form perinuclear aggregates and enhanced cell death-inducing activity of MblC overexpression.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Valencia, Valencia, Spain.

ABSTRACT

Background: Muscleblind-like proteins (MBNL) have been involved in a developmental switch in the use of defined cassette exons. Such transition fails in the CTG repeat expansion disease myotonic dystrophy due, in part, to sequestration of MBNL proteins by CUG repeat RNA. Four protein isoforms (MblA-D) are coded by the unique Drosophila muscleblind gene.

Methodology/principal findings: We used evolutionary, genetic and cell culture approaches to study muscleblind (mbl) function in flies. The evolutionary study showed that the MblC protein isoform was readily conserved from nematods to Drosophila, which suggests that it performs the most ancestral muscleblind functions. Overexpression of MblC in the fly eye precursors led to an externally rough eye morphology. This phenotype was used in a genetic screen to identify five dominant suppressors and 13 dominant enhancers including Drosophila CUG-BP1 homolog aret, exon junction complex components tsunagi and Aly, and pro-apoptotic genes Traf1 and reaper. We further investigated Muscleblind implication in apoptosis and splicing regulation. We found missplicing of troponin T in muscleblind mutant pupae and confirmed Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3) minigene splicing in human HEK cells. MblC overexpression in the wing imaginal disc activated apoptosis in a spatially restricted manner. Bioinformatics analysis identified a conserved FKRP motif, weakly resembling a sumoylation target site, in the MblC-specific sequence. Site-directed mutagenesis of the motif revealed no change in activity of mutant MblC on TnnT3 minigene splicing or aberrant binding to CUG repeat RNA, but altered the ability of the protein to form perinuclear aggregates and enhanced cell death-inducing activity of MblC overexpression.

Conclusions/significance: Taken together our genetic approach identify cellular processes influenced by Muscleblind function, whereas in vivo and cell culture experiments define Drosophila troponin T as a new Muscleblind target, reveal a potential involvement of MblC in programmed cell death and recognize the FKRP motif as a putative regulator of MblC function and/or subcellular location in the cell.

Show MeSH
Related in: MedlinePlus