Limits...
Genetic Interactions between the Members of the SMN-Gemins Complex in Drosophila.

Borg RM, Bordonne R, Vassallo N, Cauchi RJ - PLoS ONE (2015)

Bottom Line: Despite multiple genetic studies, the Gemin proteins have not been identified as prominent modifiers of SMN-associated mutant phenotypes.We show a modifier effect by all three members of the minimalistic fly SMN-Gemins complex within the muscle compartment of the motor unit.The toxicity associated with increased Gemin2 levels is conserved in the yeast S. pombe in which we find that the cytoplasmic retention of Sm proteins, likely reflecting a block in the snRNP assembly pathway, is a contributing factor.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biochemistry, Faculty of Medicine and Surgery, University of Malta, Msida, Malta GC; Institut de Génétique Moléculaire de Montpellier, CNRS-UMR5535, Université Montpellier 1 and 2, Montpellier, France.

ABSTRACT
The SMN-Gemins complex is composed of Gemins 2-8, Unrip and the survival motor neuron (SMN) protein. Limiting levels of SMN result in the neuromuscular disorder, spinal muscular atrophy (SMA), which is presently untreatable. The most-documented function of the SMN-Gemins complex concerns the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). Despite multiple genetic studies, the Gemin proteins have not been identified as prominent modifiers of SMN-associated mutant phenotypes. In the present report, we make use of the Drosophila model organism to investigate whether viability and motor phenotypes associated with a hypomorphic Gemin3 mutant are enhanced by changes in the levels of SMN, Gemin2 and Gemin5 brought about by various genetic manipulations. We show a modifier effect by all three members of the minimalistic fly SMN-Gemins complex within the muscle compartment of the motor unit. Interestingly, muscle-specific overexpression of Gemin2 was by itself sufficient to depress normal motor function and its enhanced upregulation in all tissues leads to a decline in fly viability. The toxicity associated with increased Gemin2 levels is conserved in the yeast S. pombe in which we find that the cytoplasmic retention of Sm proteins, likely reflecting a block in the snRNP assembly pathway, is a contributing factor. We propose that a disruption in the normal stoichiometry of the SMN-Gemins complex depresses its function with consequences that are detrimental to the motor system.

No MeSH data available.


Related in: MedlinePlus

Overexpression of full-length or N-terminal truncated Gemin5 escalate flight and viability defects of a Gemin3 hypomorphic mutant.(A) A double but not a single dose of Gemin3 hypomorph, Gem3BART, driven via the pan-muscular driver Mef2-GAL4 driver results in a high percentage of adult flies that are flight-impaired at all the time points measured (left panel). Overexpression of either full-length (middle panel) or N-terminally truncated Gemin5 (right panel) enhance the flight defects associated with the ectopic expression of Gem3BART in muscle tissues. Flight performance was determined via Droso-Drome runs, in which the height a fly falls indicates its flight capability. Fliers concentrate in the upper sectors whereas non-fliers drop to sector 1, the lowest sector. (B) Percentage number of flies alive assessed at different time points during adulthood. Muscle-restricted ectopic expression of two Gem3BART transgenes results in a statistically significant drop in adult fly survival compared to the expression of a single transgene. A similar decline in survival is obtained on overexpression of either full-length or N-terminally truncated Gemin5 in a Gem3BART background. In both (A) and (B) data presented are the mean ± S.E.M. of at least 4 independent experiments, and n ≥ 60 per genotype for each time point measured. Significance was tested by the unpaired t-test and two-way ANOVA in (A) and (B), respectively, and for all data, **p<0.01, ***p<0.001, and ****p<0.0001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4476591&req=5

pone.0130974.g003: Overexpression of full-length or N-terminal truncated Gemin5 escalate flight and viability defects of a Gemin3 hypomorphic mutant.(A) A double but not a single dose of Gemin3 hypomorph, Gem3BART, driven via the pan-muscular driver Mef2-GAL4 driver results in a high percentage of adult flies that are flight-impaired at all the time points measured (left panel). Overexpression of either full-length (middle panel) or N-terminally truncated Gemin5 (right panel) enhance the flight defects associated with the ectopic expression of Gem3BART in muscle tissues. Flight performance was determined via Droso-Drome runs, in which the height a fly falls indicates its flight capability. Fliers concentrate in the upper sectors whereas non-fliers drop to sector 1, the lowest sector. (B) Percentage number of flies alive assessed at different time points during adulthood. Muscle-restricted ectopic expression of two Gem3BART transgenes results in a statistically significant drop in adult fly survival compared to the expression of a single transgene. A similar decline in survival is obtained on overexpression of either full-length or N-terminally truncated Gemin5 in a Gem3BART background. In both (A) and (B) data presented are the mean ± S.E.M. of at least 4 independent experiments, and n ≥ 60 per genotype for each time point measured. Significance was tested by the unpaired t-test and two-way ANOVA in (A) and (B), respectively, and for all data, **p<0.01, ***p<0.001, and ****p<0.0001.

Mentions: Pan-muscular overexpression of Gem3∆N, a truncated Gemin3 mutant lacking the helicase core, results in reduced viability, motor defects and flight muscle atrophy [25]. Recently, based on genetic evidence, we demonstrated that the Gem3∆N mutant mimics a loss-of-function by presumably interfering at some level with the activity of the endogenous Gemin3 protein or its associated complex [37]. In this study, we screened a collection of randomly-inserted Gem3∆N lines and identified strains in which activation of Gem3∆N gene via the UAS/GAL4 system results in the induction of only low levels of the mutant. One particular transgene, Gem3BART, was selected for subsequent experiments. Expression of this hypomorph in muscular tissue starting early during development (Mef2-GAL4>Gem3BART) has no effect on both motor function and survival throughout adulthood (Fig 3). Motor function was measured via a flight assay in which the height a fly falls in a cylinder determines its flight performance. Hence, fliers are capable of holding onto the walls of upper sectors whereas flight-defective organisms drop to lower sectors. It is noteworthy that increasing the dose of Gem3BART has drastic consequences. Indeed, flies with two copies of the mutant (Mef2-GAL4>Gem3BARTX2) are flightless in their majority on day 5 post-eclosion, the earliest time point measured (Fig 3A). In this regard, 90% of the flies fall straight to the lowest sector. Flight ability declines rapidly so that on the remaining time points starting from day 15, all flies assayed were flight defective. Compared with flies having only one copy of Gem3BART, those with a double dose were found to have an age-dependent progressive decline in adult survival (Fig 3B).


Genetic Interactions between the Members of the SMN-Gemins Complex in Drosophila.

Borg RM, Bordonne R, Vassallo N, Cauchi RJ - PLoS ONE (2015)

Overexpression of full-length or N-terminal truncated Gemin5 escalate flight and viability defects of a Gemin3 hypomorphic mutant.(A) A double but not a single dose of Gemin3 hypomorph, Gem3BART, driven via the pan-muscular driver Mef2-GAL4 driver results in a high percentage of adult flies that are flight-impaired at all the time points measured (left panel). Overexpression of either full-length (middle panel) or N-terminally truncated Gemin5 (right panel) enhance the flight defects associated with the ectopic expression of Gem3BART in muscle tissues. Flight performance was determined via Droso-Drome runs, in which the height a fly falls indicates its flight capability. Fliers concentrate in the upper sectors whereas non-fliers drop to sector 1, the lowest sector. (B) Percentage number of flies alive assessed at different time points during adulthood. Muscle-restricted ectopic expression of two Gem3BART transgenes results in a statistically significant drop in adult fly survival compared to the expression of a single transgene. A similar decline in survival is obtained on overexpression of either full-length or N-terminally truncated Gemin5 in a Gem3BART background. In both (A) and (B) data presented are the mean ± S.E.M. of at least 4 independent experiments, and n ≥ 60 per genotype for each time point measured. Significance was tested by the unpaired t-test and two-way ANOVA in (A) and (B), respectively, and for all data, **p<0.01, ***p<0.001, and ****p<0.0001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476591&req=5

pone.0130974.g003: Overexpression of full-length or N-terminal truncated Gemin5 escalate flight and viability defects of a Gemin3 hypomorphic mutant.(A) A double but not a single dose of Gemin3 hypomorph, Gem3BART, driven via the pan-muscular driver Mef2-GAL4 driver results in a high percentage of adult flies that are flight-impaired at all the time points measured (left panel). Overexpression of either full-length (middle panel) or N-terminally truncated Gemin5 (right panel) enhance the flight defects associated with the ectopic expression of Gem3BART in muscle tissues. Flight performance was determined via Droso-Drome runs, in which the height a fly falls indicates its flight capability. Fliers concentrate in the upper sectors whereas non-fliers drop to sector 1, the lowest sector. (B) Percentage number of flies alive assessed at different time points during adulthood. Muscle-restricted ectopic expression of two Gem3BART transgenes results in a statistically significant drop in adult fly survival compared to the expression of a single transgene. A similar decline in survival is obtained on overexpression of either full-length or N-terminally truncated Gemin5 in a Gem3BART background. In both (A) and (B) data presented are the mean ± S.E.M. of at least 4 independent experiments, and n ≥ 60 per genotype for each time point measured. Significance was tested by the unpaired t-test and two-way ANOVA in (A) and (B), respectively, and for all data, **p<0.01, ***p<0.001, and ****p<0.0001.
Mentions: Pan-muscular overexpression of Gem3∆N, a truncated Gemin3 mutant lacking the helicase core, results in reduced viability, motor defects and flight muscle atrophy [25]. Recently, based on genetic evidence, we demonstrated that the Gem3∆N mutant mimics a loss-of-function by presumably interfering at some level with the activity of the endogenous Gemin3 protein or its associated complex [37]. In this study, we screened a collection of randomly-inserted Gem3∆N lines and identified strains in which activation of Gem3∆N gene via the UAS/GAL4 system results in the induction of only low levels of the mutant. One particular transgene, Gem3BART, was selected for subsequent experiments. Expression of this hypomorph in muscular tissue starting early during development (Mef2-GAL4>Gem3BART) has no effect on both motor function and survival throughout adulthood (Fig 3). Motor function was measured via a flight assay in which the height a fly falls in a cylinder determines its flight performance. Hence, fliers are capable of holding onto the walls of upper sectors whereas flight-defective organisms drop to lower sectors. It is noteworthy that increasing the dose of Gem3BART has drastic consequences. Indeed, flies with two copies of the mutant (Mef2-GAL4>Gem3BARTX2) are flightless in their majority on day 5 post-eclosion, the earliest time point measured (Fig 3A). In this regard, 90% of the flies fall straight to the lowest sector. Flight ability declines rapidly so that on the remaining time points starting from day 15, all flies assayed were flight defective. Compared with flies having only one copy of Gem3BART, those with a double dose were found to have an age-dependent progressive decline in adult survival (Fig 3B).

Bottom Line: Despite multiple genetic studies, the Gemin proteins have not been identified as prominent modifiers of SMN-associated mutant phenotypes.We show a modifier effect by all three members of the minimalistic fly SMN-Gemins complex within the muscle compartment of the motor unit.The toxicity associated with increased Gemin2 levels is conserved in the yeast S. pombe in which we find that the cytoplasmic retention of Sm proteins, likely reflecting a block in the snRNP assembly pathway, is a contributing factor.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biochemistry, Faculty of Medicine and Surgery, University of Malta, Msida, Malta GC; Institut de Génétique Moléculaire de Montpellier, CNRS-UMR5535, Université Montpellier 1 and 2, Montpellier, France.

ABSTRACT
The SMN-Gemins complex is composed of Gemins 2-8, Unrip and the survival motor neuron (SMN) protein. Limiting levels of SMN result in the neuromuscular disorder, spinal muscular atrophy (SMA), which is presently untreatable. The most-documented function of the SMN-Gemins complex concerns the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). Despite multiple genetic studies, the Gemin proteins have not been identified as prominent modifiers of SMN-associated mutant phenotypes. In the present report, we make use of the Drosophila model organism to investigate whether viability and motor phenotypes associated with a hypomorphic Gemin3 mutant are enhanced by changes in the levels of SMN, Gemin2 and Gemin5 brought about by various genetic manipulations. We show a modifier effect by all three members of the minimalistic fly SMN-Gemins complex within the muscle compartment of the motor unit. Interestingly, muscle-specific overexpression of Gemin2 was by itself sufficient to depress normal motor function and its enhanced upregulation in all tissues leads to a decline in fly viability. The toxicity associated with increased Gemin2 levels is conserved in the yeast S. pombe in which we find that the cytoplasmic retention of Sm proteins, likely reflecting a block in the snRNP assembly pathway, is a contributing factor. We propose that a disruption in the normal stoichiometry of the SMN-Gemins complex depresses its function with consequences that are detrimental to the motor system.

No MeSH data available.


Related in: MedlinePlus