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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

Augmentation or attenuation of SMN levels expose the motor and viability defects associated with the Gem3BART hypomorph.(A) Left panel: Overexpression of full-length SMN (GFP-SmnFL) or a version lacking the region hosting the YG box (GFP-Smn∆6) in muscle tissue is by itself inconsequential. However, in combination with Gem3BART, flies exhibit either lethality (Mef2-GAL4>Gem3BART+ GFP-SmnFL) or flightlessness (Mef2-GAL4>Gem3BART+ GFP-Smn∆6). Statistical significance was determined for differences between the Mef2-GAL4>Gem3BART+ GFP-Smn∆6 genotype, and the control Mef2-GAL4>GFP-Smn∆6 genotype. Middle panel: Transgenic increase (Mef2-GAL4>Flag-SmnFL) or decrease (Mef2-GAL4>Smn-IRFL26B) in SMN levels, alone, has no impact on motor behaviour whereas in combination with Gem3BART, the end-result is flight impairment (Mef2-GAL4>Gem3BART+ Flag-SmnFL) and lethality (Mef2-GAL4>Gem3BART+ Smn-IRFL26B), respectively. Statistical significance was determined for differences between the Mef2-GAL4>Gem3BART+ Flag-SmnFL genotype, and the control Mef2-GAL4>Flag-SmnFL genotype. Right panel: Smn knockdown, alone, through either targeting the N-terminus (Mef2-GAL4>Smn-IRN4) or the C-terminus (Mef2-GAL4>Smn-IRC24) has no obvious effect on flight behaviour, but it enhances the Gem3BART phenotype leading to lethality in Mef2-GAL4>Gem3BART+ Smn-IRN4 flies or flight defects in Mef2-GAL4>Gem3BART+ Smn-IRC24 flies. Statistical significance was determined for differences between the Mef2-GAL4>Gem3BART+ Smn-IRC24 genotype, and the control Mef2-GAL4>Smn-IRC24 genotype. (B) Compared to controls, a statistically significant drop in adult viability throughout adulthood is observed in flies with pan-muscular ectopic expression of Gem3BART and either C-terminal targeted SMN knockdown (Mef2-GAL4>Gem3BART+ Smn-IRC24) or overexpression of the SMN∆6 variant (Mef2-GAL4>Gem3BART+ GFP-Smn∆6). In both (A) and (B) data presented are the mean ± S.E.M. of at least 4 independent experiments, and n ≥ 100 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<0.05, and ****p<0.0001.
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pone.0130974.g005: Augmentation or attenuation of SMN levels expose the motor and viability defects associated with the Gem3BART hypomorph.(A) Left panel: Overexpression of full-length SMN (GFP-SmnFL) or a version lacking the region hosting the YG box (GFP-Smn∆6) in muscle tissue is by itself inconsequential. However, in combination with Gem3BART, flies exhibit either lethality (Mef2-GAL4>Gem3BART+ GFP-SmnFL) or flightlessness (Mef2-GAL4>Gem3BART+ GFP-Smn∆6). Statistical significance was determined for differences between the Mef2-GAL4>Gem3BART+ GFP-Smn∆6 genotype, and the control Mef2-GAL4>GFP-Smn∆6 genotype. Middle panel: Transgenic increase (Mef2-GAL4>Flag-SmnFL) or decrease (Mef2-GAL4>Smn-IRFL26B) in SMN levels, alone, has no impact on motor behaviour whereas in combination with Gem3BART, the end-result is flight impairment (Mef2-GAL4>Gem3BART+ Flag-SmnFL) and lethality (Mef2-GAL4>Gem3BART+ Smn-IRFL26B), respectively. Statistical significance was determined for differences between the Mef2-GAL4>Gem3BART+ Flag-SmnFL genotype, and the control Mef2-GAL4>Flag-SmnFL genotype. Right panel: Smn knockdown, alone, through either targeting the N-terminus (Mef2-GAL4>Smn-IRN4) or the C-terminus (Mef2-GAL4>Smn-IRC24) has no obvious effect on flight behaviour, but it enhances the Gem3BART phenotype leading to lethality in Mef2-GAL4>Gem3BART+ Smn-IRN4 flies or flight defects in Mef2-GAL4>Gem3BART+ Smn-IRC24 flies. Statistical significance was determined for differences between the Mef2-GAL4>Gem3BART+ Smn-IRC24 genotype, and the control Mef2-GAL4>Smn-IRC24 genotype. (B) Compared to controls, a statistically significant drop in adult viability throughout adulthood is observed in flies with pan-muscular ectopic expression of Gem3BART and either C-terminal targeted SMN knockdown (Mef2-GAL4>Gem3BART+ Smn-IRC24) or overexpression of the SMN∆6 variant (Mef2-GAL4>Gem3BART+ GFP-Smn∆6). In both (A) and (B) data presented are the mean ± S.E.M. of at least 4 independent experiments, and n ≥ 100 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<0.05, and ****p<0.0001.

Mentions: In Drosophila, Grice and Liu [49] reported that although SMN overexpression does not influence viability, it affects development leading to an alteration in both brain growth and the timing of cell differentiation in the testis. We asked whether the upregulation of SMN in muscle tissue has an effect on motor behaviour and, to this end, we found none whatsoever using two commonly used full-length Smn transgenes (GFP-SmnFL and Flag-SmnFL) [22, 23] or a version lacking the region hosting the YG box domain (GFP-Smn∆6) [22], a highly-conserved domain required for SMN oligomerisation (reviewed in [1]) (Fig 5A). However, in combination with Gem3BART, a surplus of SMN results in lethality with few escapers (Mef-GAL4> Gem3BART+ Flag-SmnFL) exhibiting severe flight defects (Fig 5A). Interestingly, overexpression of SMN∆6 had a lesser impact, hence, flies expressing both SMN∆6 and Gem3BART in muscle are developmentally viable but in their majority they are flightless. Observation of adult flies with this genotype (Mef-GAL4>Gem3BART+ GFP-Smn∆6) shows that compared to controls they experience a significant decline in survival throughout adulthood (Fig 5B).


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

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

Augmentation or attenuation of SMN levels expose the motor and viability defects associated with the Gem3BART hypomorph.(A) Left panel: Overexpression of full-length SMN (GFP-SmnFL) or a version lacking the region hosting the YG box (GFP-Smn∆6) in muscle tissue is by itself inconsequential. However, in combination with Gem3BART, flies exhibit either lethality (Mef2-GAL4>Gem3BART+ GFP-SmnFL) or flightlessness (Mef2-GAL4>Gem3BART+ GFP-Smn∆6). Statistical significance was determined for differences between the Mef2-GAL4>Gem3BART+ GFP-Smn∆6 genotype, and the control Mef2-GAL4>GFP-Smn∆6 genotype. Middle panel: Transgenic increase (Mef2-GAL4>Flag-SmnFL) or decrease (Mef2-GAL4>Smn-IRFL26B) in SMN levels, alone, has no impact on motor behaviour whereas in combination with Gem3BART, the end-result is flight impairment (Mef2-GAL4>Gem3BART+ Flag-SmnFL) and lethality (Mef2-GAL4>Gem3BART+ Smn-IRFL26B), respectively. Statistical significance was determined for differences between the Mef2-GAL4>Gem3BART+ Flag-SmnFL genotype, and the control Mef2-GAL4>Flag-SmnFL genotype. Right panel: Smn knockdown, alone, through either targeting the N-terminus (Mef2-GAL4>Smn-IRN4) or the C-terminus (Mef2-GAL4>Smn-IRC24) has no obvious effect on flight behaviour, but it enhances the Gem3BART phenotype leading to lethality in Mef2-GAL4>Gem3BART+ Smn-IRN4 flies or flight defects in Mef2-GAL4>Gem3BART+ Smn-IRC24 flies. Statistical significance was determined for differences between the Mef2-GAL4>Gem3BART+ Smn-IRC24 genotype, and the control Mef2-GAL4>Smn-IRC24 genotype. (B) Compared to controls, a statistically significant drop in adult viability throughout adulthood is observed in flies with pan-muscular ectopic expression of Gem3BART and either C-terminal targeted SMN knockdown (Mef2-GAL4>Gem3BART+ Smn-IRC24) or overexpression of the SMN∆6 variant (Mef2-GAL4>Gem3BART+ GFP-Smn∆6). In both (A) and (B) data presented are the mean ± S.E.M. of at least 4 independent experiments, and n ≥ 100 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<0.05, and ****p<0.0001.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130974.g005: Augmentation or attenuation of SMN levels expose the motor and viability defects associated with the Gem3BART hypomorph.(A) Left panel: Overexpression of full-length SMN (GFP-SmnFL) or a version lacking the region hosting the YG box (GFP-Smn∆6) in muscle tissue is by itself inconsequential. However, in combination with Gem3BART, flies exhibit either lethality (Mef2-GAL4>Gem3BART+ GFP-SmnFL) or flightlessness (Mef2-GAL4>Gem3BART+ GFP-Smn∆6). Statistical significance was determined for differences between the Mef2-GAL4>Gem3BART+ GFP-Smn∆6 genotype, and the control Mef2-GAL4>GFP-Smn∆6 genotype. Middle panel: Transgenic increase (Mef2-GAL4>Flag-SmnFL) or decrease (Mef2-GAL4>Smn-IRFL26B) in SMN levels, alone, has no impact on motor behaviour whereas in combination with Gem3BART, the end-result is flight impairment (Mef2-GAL4>Gem3BART+ Flag-SmnFL) and lethality (Mef2-GAL4>Gem3BART+ Smn-IRFL26B), respectively. Statistical significance was determined for differences between the Mef2-GAL4>Gem3BART+ Flag-SmnFL genotype, and the control Mef2-GAL4>Flag-SmnFL genotype. Right panel: Smn knockdown, alone, through either targeting the N-terminus (Mef2-GAL4>Smn-IRN4) or the C-terminus (Mef2-GAL4>Smn-IRC24) has no obvious effect on flight behaviour, but it enhances the Gem3BART phenotype leading to lethality in Mef2-GAL4>Gem3BART+ Smn-IRN4 flies or flight defects in Mef2-GAL4>Gem3BART+ Smn-IRC24 flies. Statistical significance was determined for differences between the Mef2-GAL4>Gem3BART+ Smn-IRC24 genotype, and the control Mef2-GAL4>Smn-IRC24 genotype. (B) Compared to controls, a statistically significant drop in adult viability throughout adulthood is observed in flies with pan-muscular ectopic expression of Gem3BART and either C-terminal targeted SMN knockdown (Mef2-GAL4>Gem3BART+ Smn-IRC24) or overexpression of the SMN∆6 variant (Mef2-GAL4>Gem3BART+ GFP-Smn∆6). In both (A) and (B) data presented are the mean ± S.E.M. of at least 4 independent experiments, and n ≥ 100 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<0.05, and ****p<0.0001.
Mentions: In Drosophila, Grice and Liu [49] reported that although SMN overexpression does not influence viability, it affects development leading to an alteration in both brain growth and the timing of cell differentiation in the testis. We asked whether the upregulation of SMN in muscle tissue has an effect on motor behaviour and, to this end, we found none whatsoever using two commonly used full-length Smn transgenes (GFP-SmnFL and Flag-SmnFL) [22, 23] or a version lacking the region hosting the YG box domain (GFP-Smn∆6) [22], a highly-conserved domain required for SMN oligomerisation (reviewed in [1]) (Fig 5A). However, in combination with Gem3BART, a surplus of SMN results in lethality with few escapers (Mef-GAL4> Gem3BART+ Flag-SmnFL) exhibiting severe flight defects (Fig 5A). Interestingly, overexpression of SMN∆6 had a lesser impact, hence, flies expressing both SMN∆6 and Gem3BART in muscle are developmentally viable but in their majority they are flightless. Observation of adult flies with this genotype (Mef-GAL4>Gem3BART+ GFP-Smn∆6) shows that compared to controls they experience a significant decline in survival throughout adulthood (Fig 5B).

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