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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

In combination with Gem3BART, Gemin5 knockdown triggers lethality whereas a reduction in the gene copy number of Gemin5 provokes impaired flight.(A) Pan-muscular Gemin5 knockdown alone has no effect on flight performance (left panel). In a heterozygous Gemin5 deficient background brought about by a chromosomal deletion (Df(2R)exu1), the hypomorphic Gem3BART motor phenotype becomes apparent on the day 5 time point and intensifies with age (middle panel). In this respect, the percentage of non-fliers in sector 1 increases significantly with age. In the heterozygous state, Smn (SmnX7) or Gemin2 deletion (Df(3L)ED4782) has no negative influence on Gem3BART (right panel). (B) Adult viability is significantly negatively impacted when a Gemin5 chromosomal deletion is coupled with Gem3BART. Gemin5 knockdown alone or Gem3BART in combination with SmnX7 induce only a mild decrease in viability throughout adulthood. Compared to these genotypes, a moderate (but not severe) decrease in adult viability is seen in flies with both Gemin3BART and a chromosomal deletion that eliminates Gemin2 (Df(3L)ED4782). 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<0.05, **p<0.01, and ****p<0.0001.
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pone.0130974.g004: In combination with Gem3BART, Gemin5 knockdown triggers lethality whereas a reduction in the gene copy number of Gemin5 provokes impaired flight.(A) Pan-muscular Gemin5 knockdown alone has no effect on flight performance (left panel). In a heterozygous Gemin5 deficient background brought about by a chromosomal deletion (Df(2R)exu1), the hypomorphic Gem3BART motor phenotype becomes apparent on the day 5 time point and intensifies with age (middle panel). In this respect, the percentage of non-fliers in sector 1 increases significantly with age. In the heterozygous state, Smn (SmnX7) or Gemin2 deletion (Df(3L)ED4782) has no negative influence on Gem3BART (right panel). (B) Adult viability is significantly negatively impacted when a Gemin5 chromosomal deletion is coupled with Gem3BART. Gemin5 knockdown alone or Gem3BART in combination with SmnX7 induce only a mild decrease in viability throughout adulthood. Compared to these genotypes, a moderate (but not severe) decrease in adult viability is seen in flies with both Gemin3BART and a chromosomal deletion that eliminates Gemin2 (Df(3L)ED4782). 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<0.05, **p<0.01, and ****p<0.0001.

Mentions: We next questioned whether the Gem3BART phenotype would also be enhanced if Gemin5 levels were reduced. Augmented Dicer-2 levels were reported to enhance Gemin5 knockdown leading to phenotypic consequences [37]. However, in the absence of elevated Dicer-2 levels, Gemin5 knockdown in muscles (Mef2-GAL4>Gem5-IRnan+sac) is uneventful (Fig 4). Importantly, flies with a pan-muscular Gemin5 knockdown coupled with the ectopic expression of Gem3BART (Mef2-GAL4>Gem3BART+ Gem5-IRnan+sac) were not adult viable. A subtler reduction in Gemin5 levels through a reduction in gene copy number via a chromosomal deletion (Df(2R)exu1) was sufficient to expose the motor and viability defects intrinsic to the Gem3BART hypomorph. In this respect, muscle-specific Gem3BART expression in a heterozygous Gemin5 deficient background (Mef2-GAL4>Gem3BART+ Df(2R)exu1) gave rise to an age-progressive decline in flight ability starting from day 5 post-eclosion (Fig 4A). Viability was also significantly affected (Fig 4B). In contrast to what we observed for Gemin5, a background with a heterozygous deficiency of either Smn (Mef2-GAL4>Gem3BART+ SmnX7) or Gemin2 (Mef2-GAL4>Gem3BART+ Df(3L)ED4782) does not enhance the Gem3BART hypomorphic phenotype. Overall, these findings are suggestive of a genetic interaction between Gemin3 and Gemin5.


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

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

In combination with Gem3BART, Gemin5 knockdown triggers lethality whereas a reduction in the gene copy number of Gemin5 provokes impaired flight.(A) Pan-muscular Gemin5 knockdown alone has no effect on flight performance (left panel). In a heterozygous Gemin5 deficient background brought about by a chromosomal deletion (Df(2R)exu1), the hypomorphic Gem3BART motor phenotype becomes apparent on the day 5 time point and intensifies with age (middle panel). In this respect, the percentage of non-fliers in sector 1 increases significantly with age. In the heterozygous state, Smn (SmnX7) or Gemin2 deletion (Df(3L)ED4782) has no negative influence on Gem3BART (right panel). (B) Adult viability is significantly negatively impacted when a Gemin5 chromosomal deletion is coupled with Gem3BART. Gemin5 knockdown alone or Gem3BART in combination with SmnX7 induce only a mild decrease in viability throughout adulthood. Compared to these genotypes, a moderate (but not severe) decrease in adult viability is seen in flies with both Gemin3BART and a chromosomal deletion that eliminates Gemin2 (Df(3L)ED4782). 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<0.05, **p<0.01, and ****p<0.0001.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130974.g004: In combination with Gem3BART, Gemin5 knockdown triggers lethality whereas a reduction in the gene copy number of Gemin5 provokes impaired flight.(A) Pan-muscular Gemin5 knockdown alone has no effect on flight performance (left panel). In a heterozygous Gemin5 deficient background brought about by a chromosomal deletion (Df(2R)exu1), the hypomorphic Gem3BART motor phenotype becomes apparent on the day 5 time point and intensifies with age (middle panel). In this respect, the percentage of non-fliers in sector 1 increases significantly with age. In the heterozygous state, Smn (SmnX7) or Gemin2 deletion (Df(3L)ED4782) has no negative influence on Gem3BART (right panel). (B) Adult viability is significantly negatively impacted when a Gemin5 chromosomal deletion is coupled with Gem3BART. Gemin5 knockdown alone or Gem3BART in combination with SmnX7 induce only a mild decrease in viability throughout adulthood. Compared to these genotypes, a moderate (but not severe) decrease in adult viability is seen in flies with both Gemin3BART and a chromosomal deletion that eliminates Gemin2 (Df(3L)ED4782). 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<0.05, **p<0.01, and ****p<0.0001.
Mentions: We next questioned whether the Gem3BART phenotype would also be enhanced if Gemin5 levels were reduced. Augmented Dicer-2 levels were reported to enhance Gemin5 knockdown leading to phenotypic consequences [37]. However, in the absence of elevated Dicer-2 levels, Gemin5 knockdown in muscles (Mef2-GAL4>Gem5-IRnan+sac) is uneventful (Fig 4). Importantly, flies with a pan-muscular Gemin5 knockdown coupled with the ectopic expression of Gem3BART (Mef2-GAL4>Gem3BART+ Gem5-IRnan+sac) were not adult viable. A subtler reduction in Gemin5 levels through a reduction in gene copy number via a chromosomal deletion (Df(2R)exu1) was sufficient to expose the motor and viability defects intrinsic to the Gem3BART hypomorph. In this respect, muscle-specific Gem3BART expression in a heterozygous Gemin5 deficient background (Mef2-GAL4>Gem3BART+ Df(2R)exu1) gave rise to an age-progressive decline in flight ability starting from day 5 post-eclosion (Fig 4A). Viability was also significantly affected (Fig 4B). In contrast to what we observed for Gemin5, a background with a heterozygous deficiency of either Smn (Mef2-GAL4>Gem3BART+ SmnX7) or Gemin2 (Mef2-GAL4>Gem3BART+ Df(3L)ED4782) does not enhance the Gem3BART hypomorphic phenotype. Overall, these findings are suggestive of a genetic interaction between Gemin3 and Gemin5.

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