Limits...
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.

Show MeSH

Related in: MedlinePlus

Muscleblind proteins regulate troponin T alternative splicing in vivo and in cell culture.(A) Drosophila troponin T splicing characterisation in wild type (OrR); mblE27/CyO, ubi-GFP; mblK7103/CyO, ubi-GFP; and the hypomorphic allelic combination mblE27/mblk7103 early pupae. Molecular weight of DNA marker bands is shown on the left. Exon composition compatible with product size is shown on the right. Rp49 is shown as control in RT-PCR. (B) Murine TnnT3 minigene was co-transfected into HEK293T cells along with plasmids expressing Drosophila Muscleblind and Bruno proteins. +F indicates presence of the foetal exon and –F its absence. (C) Bar graph representing the average intensity of +F (light grey) and -F (dark grey) bands, as percentage of total, in three replica experiments, except for co-transfection of MblB that could only be amplified once. Statistically significant differences from vector alone controls (GFP lane) are denoted by an asterisk (p-value<0.01). Error bars are standard deviations. Bruno proteins did not significantly modify minigene alternative splicing.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2238819&req=5

pone-0001613-g002: Muscleblind proteins regulate troponin T alternative splicing in vivo and in cell culture.(A) Drosophila troponin T splicing characterisation in wild type (OrR); mblE27/CyO, ubi-GFP; mblK7103/CyO, ubi-GFP; and the hypomorphic allelic combination mblE27/mblk7103 early pupae. Molecular weight of DNA marker bands is shown on the left. Exon composition compatible with product size is shown on the right. Rp49 is shown as control in RT-PCR. (B) Murine TnnT3 minigene was co-transfected into HEK293T cells along with plasmids expressing Drosophila Muscleblind and Bruno proteins. +F indicates presence of the foetal exon and –F its absence. (C) Bar graph representing the average intensity of +F (light grey) and -F (dark grey) bands, as percentage of total, in three replica experiments, except for co-transfection of MblB that could only be amplified once. Statistically significant differences from vector alone controls (GFP lane) are denoted by an asterisk (p-value<0.01). Error bars are standard deviations. Bruno proteins did not significantly modify minigene alternative splicing.

Mentions: Mbnl1 knockout mice show splicing defects in both cardiac Troponin T (Tnnt2) and fast skeletal muscle Troponin T (TnnT3) [11]. The Drosophila genome presents a unique troponin T (tnT) gene, which was found to undergo tissue-specific alternative splicing during development [41]. Four splicing variants differ in the inclusion or exclusion of exons E3, E4 and E5. We performed RT-PCR amplifying exons E2 to E6 of tnT mRNA to detect all described splicing isoforms in muscleblind mutant embryo, pupae and adults. Embryo and adult RNA extractions showed no detectable differences. Early mutant pupae, however, showed an increment in the tnT isoform specific of tergal depressor of trochanter (TDT) and indirect flight muscles (IFM; Figure 2A) when compared to controls. The alteration was also clearly detected in muscleblind heterozygotes (mblE27/CyO, ubi-GFP), but not in hypomorphic heterozygotes (mblk7103/CyO, ubi-GFP), thus suggesting a muscleblind dose effect.


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)

Muscleblind proteins regulate troponin T alternative splicing in vivo and in cell culture.(A) Drosophila troponin T splicing characterisation in wild type (OrR); mblE27/CyO, ubi-GFP; mblK7103/CyO, ubi-GFP; and the hypomorphic allelic combination mblE27/mblk7103 early pupae. Molecular weight of DNA marker bands is shown on the left. Exon composition compatible with product size is shown on the right. Rp49 is shown as control in RT-PCR. (B) Murine TnnT3 minigene was co-transfected into HEK293T cells along with plasmids expressing Drosophila Muscleblind and Bruno proteins. +F indicates presence of the foetal exon and –F its absence. (C) Bar graph representing the average intensity of +F (light grey) and -F (dark grey) bands, as percentage of total, in three replica experiments, except for co-transfection of MblB that could only be amplified once. Statistically significant differences from vector alone controls (GFP lane) are denoted by an asterisk (p-value<0.01). Error bars are standard deviations. Bruno proteins did not significantly modify minigene alternative splicing.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2238819&req=5

pone-0001613-g002: Muscleblind proteins regulate troponin T alternative splicing in vivo and in cell culture.(A) Drosophila troponin T splicing characterisation in wild type (OrR); mblE27/CyO, ubi-GFP; mblK7103/CyO, ubi-GFP; and the hypomorphic allelic combination mblE27/mblk7103 early pupae. Molecular weight of DNA marker bands is shown on the left. Exon composition compatible with product size is shown on the right. Rp49 is shown as control in RT-PCR. (B) Murine TnnT3 minigene was co-transfected into HEK293T cells along with plasmids expressing Drosophila Muscleblind and Bruno proteins. +F indicates presence of the foetal exon and –F its absence. (C) Bar graph representing the average intensity of +F (light grey) and -F (dark grey) bands, as percentage of total, in three replica experiments, except for co-transfection of MblB that could only be amplified once. Statistically significant differences from vector alone controls (GFP lane) are denoted by an asterisk (p-value<0.01). Error bars are standard deviations. Bruno proteins did not significantly modify minigene alternative splicing.
Mentions: Mbnl1 knockout mice show splicing defects in both cardiac Troponin T (Tnnt2) and fast skeletal muscle Troponin T (TnnT3) [11]. The Drosophila genome presents a unique troponin T (tnT) gene, which was found to undergo tissue-specific alternative splicing during development [41]. Four splicing variants differ in the inclusion or exclusion of exons E3, E4 and E5. We performed RT-PCR amplifying exons E2 to E6 of tnT mRNA to detect all described splicing isoforms in muscleblind mutant embryo, pupae and adults. Embryo and adult RNA extractions showed no detectable differences. Early mutant pupae, however, showed an increment in the tnT isoform specific of tergal depressor of trochanter (TDT) and indirect flight muscles (IFM; Figure 2A) when compared to controls. The alteration was also clearly detected in muscleblind heterozygotes (mblE27/CyO, ubi-GFP), but not in hypomorphic heterozygotes (mblk7103/CyO, ubi-GFP), thus suggesting a muscleblind dose effect.

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