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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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mblC overexpression activates apoptosis in vivo, but not significantly in cell culture.Confocal micrographs of third instar wing imaginal discs from en-Gal4 mblC/+ (A,C,E) and en-Gal4/+ controls (B,D) stained with an anti-Mbl (A), anti mammalian Caspase-3 antibody (B,C), or TUNEL assay (D,E). Wing imaginal discs of en-Gal4 UAS-mblC (C) flies show activation of executioner caspase-3 in cells over-expressing MblC (A) in the posterior compartment where the en-Gal4 driver is active. Despite the fact that MblC overexpression levels are similar in posterior pouch (A, bent arrow) and notum cells (A, arrowhead), caspase-3 is not detected activated in prospective notum cells (C). A TUNEL assay to detect DNA fragmentation that results from apoptosis signalling cascades reproduced the same pattern of apoptotic cells (D,E) detected by caspase-3 activation. (F) Bar graph representing the average number (from quadruplicates) of live cells 48 h after transfection of plasmids expressing the indicated Muscleblind protein isoforms. Overexpression of Muscleblind isoforms did not significantly reduce Drosophila S2 cell viability in cell culture conditions. Error bars are standard deviations. (G) Western blot of protein extracts from S2 cells transfected as in (F) with the indicated Muscleblind proteins and detected with an anti-Muscleblind antibody [47]. Lower molecular weight bands in lanes MblA and MblB are degradation products. MblD could not be detected by western blotting. Predicted molecular weights are: MblA, 22.65 kDa; MblB, 34.46 kDa; MblC 26.91 kDa.
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pone-0001613-g003: mblC overexpression activates apoptosis in vivo, but not significantly in cell culture.Confocal micrographs of third instar wing imaginal discs from en-Gal4 mblC/+ (A,C,E) and en-Gal4/+ controls (B,D) stained with an anti-Mbl (A), anti mammalian Caspase-3 antibody (B,C), or TUNEL assay (D,E). Wing imaginal discs of en-Gal4 UAS-mblC (C) flies show activation of executioner caspase-3 in cells over-expressing MblC (A) in the posterior compartment where the en-Gal4 driver is active. Despite the fact that MblC overexpression levels are similar in posterior pouch (A, bent arrow) and notum cells (A, arrowhead), caspase-3 is not detected activated in prospective notum cells (C). A TUNEL assay to detect DNA fragmentation that results from apoptosis signalling cascades reproduced the same pattern of apoptotic cells (D,E) detected by caspase-3 activation. (F) Bar graph representing the average number (from quadruplicates) of live cells 48 h after transfection of plasmids expressing the indicated Muscleblind protein isoforms. Overexpression of Muscleblind isoforms did not significantly reduce Drosophila S2 cell viability in cell culture conditions. Error bars are standard deviations. (G) Western blot of protein extracts from S2 cells transfected as in (F) with the indicated Muscleblind proteins and detected with an anti-Muscleblind antibody [47]. Lower molecular weight bands in lanes MblA and MblB are degradation products. MblD could not be detected by western blotting. Predicted molecular weights are: MblA, 22.65 kDa; MblB, 34.46 kDa; MblC 26.91 kDa.

Mentions: Genetic interactions with key regulators of apoptosis prompted the possibility that muscleblind could direct or indirectly participate in the apoptotic process. In order to confirm this possibility in vivo, we first analyzed the phenotype brought about by overexpression of mblC in the posterior compartment of the wing imaginal disc (en-Gal4>UAS-mblC). Lack of laminar tissue could originate from reduced proliferation of disc cells or from an excess of cell death (Figure 1J). Immunostaining with anti mammalian Caspase-3 antibody showed a robust activation of caspase-3 in cells overexpressing MblC under the control of the en-Gal4 driver (Figure 3A–C), decapentaplegic-Gal4 or patched-Gal4 (not shown). However, not all cells overexpressing MblC showed the same susceptibility to caspase cleavage. Within the fate map of the wing imaginal disc, whereas posterior notum or ventral pleura cells did not significantly promote caspase-3 activation, posterior wing margin and pouch cells strongly activated caspase 3. In order to confirm that caspase activation was due to the activation of the apoptotic pathway, and not to other functions described for caspases (see [42] for examples), we used terminal transferase dUTP nick end labeling (TUNEL) to detect DNA fragmentation that results from apoptotic signaling cascades. Using this assay we detected several apoptotic cells in the posterior compartment of the wing disc when en-Gal4 drove MblC overexpression. The assay also confirmed the spatially restricted susceptibility to enter apoptosis, in particular lack of apoptosis in posterior notum and pleura (Figure 3D,E).


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)

mblC overexpression activates apoptosis in vivo, but not significantly in cell culture.Confocal micrographs of third instar wing imaginal discs from en-Gal4 mblC/+ (A,C,E) and en-Gal4/+ controls (B,D) stained with an anti-Mbl (A), anti mammalian Caspase-3 antibody (B,C), or TUNEL assay (D,E). Wing imaginal discs of en-Gal4 UAS-mblC (C) flies show activation of executioner caspase-3 in cells over-expressing MblC (A) in the posterior compartment where the en-Gal4 driver is active. Despite the fact that MblC overexpression levels are similar in posterior pouch (A, bent arrow) and notum cells (A, arrowhead), caspase-3 is not detected activated in prospective notum cells (C). A TUNEL assay to detect DNA fragmentation that results from apoptosis signalling cascades reproduced the same pattern of apoptotic cells (D,E) detected by caspase-3 activation. (F) Bar graph representing the average number (from quadruplicates) of live cells 48 h after transfection of plasmids expressing the indicated Muscleblind protein isoforms. Overexpression of Muscleblind isoforms did not significantly reduce Drosophila S2 cell viability in cell culture conditions. Error bars are standard deviations. (G) Western blot of protein extracts from S2 cells transfected as in (F) with the indicated Muscleblind proteins and detected with an anti-Muscleblind antibody [47]. Lower molecular weight bands in lanes MblA and MblB are degradation products. MblD could not be detected by western blotting. Predicted molecular weights are: MblA, 22.65 kDa; MblB, 34.46 kDa; MblC 26.91 kDa.
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Related In: Results  -  Collection

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pone-0001613-g003: mblC overexpression activates apoptosis in vivo, but not significantly in cell culture.Confocal micrographs of third instar wing imaginal discs from en-Gal4 mblC/+ (A,C,E) and en-Gal4/+ controls (B,D) stained with an anti-Mbl (A), anti mammalian Caspase-3 antibody (B,C), or TUNEL assay (D,E). Wing imaginal discs of en-Gal4 UAS-mblC (C) flies show activation of executioner caspase-3 in cells over-expressing MblC (A) in the posterior compartment where the en-Gal4 driver is active. Despite the fact that MblC overexpression levels are similar in posterior pouch (A, bent arrow) and notum cells (A, arrowhead), caspase-3 is not detected activated in prospective notum cells (C). A TUNEL assay to detect DNA fragmentation that results from apoptosis signalling cascades reproduced the same pattern of apoptotic cells (D,E) detected by caspase-3 activation. (F) Bar graph representing the average number (from quadruplicates) of live cells 48 h after transfection of plasmids expressing the indicated Muscleblind protein isoforms. Overexpression of Muscleblind isoforms did not significantly reduce Drosophila S2 cell viability in cell culture conditions. Error bars are standard deviations. (G) Western blot of protein extracts from S2 cells transfected as in (F) with the indicated Muscleblind proteins and detected with an anti-Muscleblind antibody [47]. Lower molecular weight bands in lanes MblA and MblB are degradation products. MblD could not be detected by western blotting. Predicted molecular weights are: MblA, 22.65 kDa; MblB, 34.46 kDa; MblC 26.91 kDa.
Mentions: Genetic interactions with key regulators of apoptosis prompted the possibility that muscleblind could direct or indirectly participate in the apoptotic process. In order to confirm this possibility in vivo, we first analyzed the phenotype brought about by overexpression of mblC in the posterior compartment of the wing imaginal disc (en-Gal4>UAS-mblC). Lack of laminar tissue could originate from reduced proliferation of disc cells or from an excess of cell death (Figure 1J). Immunostaining with anti mammalian Caspase-3 antibody showed a robust activation of caspase-3 in cells overexpressing MblC under the control of the en-Gal4 driver (Figure 3A–C), decapentaplegic-Gal4 or patched-Gal4 (not shown). However, not all cells overexpressing MblC showed the same susceptibility to caspase cleavage. Within the fate map of the wing imaginal disc, whereas posterior notum or ventral pleura cells did not significantly promote caspase-3 activation, posterior wing margin and pouch cells strongly activated caspase 3. In order to confirm that caspase activation was due to the activation of the apoptotic pathway, and not to other functions described for caspases (see [42] for examples), we used terminal transferase dUTP nick end labeling (TUNEL) to detect DNA fragmentation that results from apoptotic signaling cascades. Using this assay we detected several apoptotic cells in the posterior compartment of the wing disc when en-Gal4 drove MblC overexpression. The assay also confirmed the spatially restricted susceptibility to enter apoptosis, in particular lack of apoptosis in posterior notum and pleura (Figure 3D,E).

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