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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 S. pombe, Gemin2 overexpression affects cell viability through retention of Sm proteins in the cytoplasm.(A) Wild-type or tdSmn cells were transformed with a plasmid carrying the S. pombe Smn gene, a plasmid carrying the S. pombe Gemin2 gene or with the empty pREP3∆ vector. Cultures of comparable density were then serially diluted, spotted on EMM2-Leu plates in the presence (expression is repressed) or absence (expression is induced) of Thiamine and incubated at 25°C for 5 days to test for their growth ability. In a wild-type or tdSmn background, growth defects are induced by upregulation of SpGem2 but not SpSMN. (B) Wild-type or tdSmn cells were transformed with a plasmid carrying GFP.SmB in combination with the plasmids indicated on the right. Cultures of comparable density were then serially diluted, spotted on EMM2-Leu-Ura- plates in the presence (expression is repressed) or absence (expression is induced) of Thiamine and incubated at 25°C for 5 days to examine their growth ability. The growth defect of either wild-type or tdSMN cells overexpressing Gemin2 is not complemented by an increase in the levels of SmB. tdSMN cells overexpressing both SmB and SpSMN, as expected, grew better than the control. However, the growth of wild-type cells with the same genetic modification was marginally inferior. (C) In GFP.SmB-expressing wild-type cells, SmB is predominantly localised to the nucleus. On upregulation of Gemin2, SmB accumulates in the cytoplasm and cells exhibit an elongated phenotype.
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pone.0130974.g008: In S. pombe, Gemin2 overexpression affects cell viability through retention of Sm proteins in the cytoplasm.(A) Wild-type or tdSmn cells were transformed with a plasmid carrying the S. pombe Smn gene, a plasmid carrying the S. pombe Gemin2 gene or with the empty pREP3∆ vector. Cultures of comparable density were then serially diluted, spotted on EMM2-Leu plates in the presence (expression is repressed) or absence (expression is induced) of Thiamine and incubated at 25°C for 5 days to test for their growth ability. In a wild-type or tdSmn background, growth defects are induced by upregulation of SpGem2 but not SpSMN. (B) Wild-type or tdSmn cells were transformed with a plasmid carrying GFP.SmB in combination with the plasmids indicated on the right. Cultures of comparable density were then serially diluted, spotted on EMM2-Leu-Ura- plates in the presence (expression is repressed) or absence (expression is induced) of Thiamine and incubated at 25°C for 5 days to examine their growth ability. The growth defect of either wild-type or tdSMN cells overexpressing Gemin2 is not complemented by an increase in the levels of SmB. tdSMN cells overexpressing both SmB and SpSMN, as expected, grew better than the control. However, the growth of wild-type cells with the same genetic modification was marginally inferior. (C) In GFP.SmB-expressing wild-type cells, SmB is predominantly localised to the nucleus. On upregulation of Gemin2, SmB accumulates in the cytoplasm and cells exhibit an elongated phenotype.

Mentions: Subsequently, we asked whether Gemin2 overexpression is also detrimental in other model organisms, and to this end, we focused on S. pombe, which has been shown to be an excellent system to model human diseases [50]. In particular, we have previously demonstrated that cells carrying a temperature-degron Smn (tdSmn) allele mimic snRNP assembly and splicing defects observed in SMN deficient metazoan cells [34]. Wild-type and tdSmn cells were transformed with a plasmid carrying SpGem2 (yip11) under the control of a very strong nmt1 promoter. Cell cultures of comparable density were subjected to a drop test to investigate their ability to grow at 25°C for 5 days. In case of the tdSmn allele, at a temperature of 25°C, the function of SMN in snRNP assembly is already disrupted [34]. Compared to control (empty plasmid), SpGem2 overexpressors displayed pronounced growth defects in either a wild-type or tdSmn background (Fig 8A). The same cannot be said for SMN. Thus, corroborating our results in Drosophila (above), overexpression of SpSmn had no negative influence on the growth rate of wild-type cells whereas, as expected, it improved growth when overexpressed in a tdSmn background.


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

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

In S. pombe, Gemin2 overexpression affects cell viability through retention of Sm proteins in the cytoplasm.(A) Wild-type or tdSmn cells were transformed with a plasmid carrying the S. pombe Smn gene, a plasmid carrying the S. pombe Gemin2 gene or with the empty pREP3∆ vector. Cultures of comparable density were then serially diluted, spotted on EMM2-Leu plates in the presence (expression is repressed) or absence (expression is induced) of Thiamine and incubated at 25°C for 5 days to test for their growth ability. In a wild-type or tdSmn background, growth defects are induced by upregulation of SpGem2 but not SpSMN. (B) Wild-type or tdSmn cells were transformed with a plasmid carrying GFP.SmB in combination with the plasmids indicated on the right. Cultures of comparable density were then serially diluted, spotted on EMM2-Leu-Ura- plates in the presence (expression is repressed) or absence (expression is induced) of Thiamine and incubated at 25°C for 5 days to examine their growth ability. The growth defect of either wild-type or tdSMN cells overexpressing Gemin2 is not complemented by an increase in the levels of SmB. tdSMN cells overexpressing both SmB and SpSMN, as expected, grew better than the control. However, the growth of wild-type cells with the same genetic modification was marginally inferior. (C) In GFP.SmB-expressing wild-type cells, SmB is predominantly localised to the nucleus. On upregulation of Gemin2, SmB accumulates in the cytoplasm and cells exhibit an elongated phenotype.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130974.g008: In S. pombe, Gemin2 overexpression affects cell viability through retention of Sm proteins in the cytoplasm.(A) Wild-type or tdSmn cells were transformed with a plasmid carrying the S. pombe Smn gene, a plasmid carrying the S. pombe Gemin2 gene or with the empty pREP3∆ vector. Cultures of comparable density were then serially diluted, spotted on EMM2-Leu plates in the presence (expression is repressed) or absence (expression is induced) of Thiamine and incubated at 25°C for 5 days to test for their growth ability. In a wild-type or tdSmn background, growth defects are induced by upregulation of SpGem2 but not SpSMN. (B) Wild-type or tdSmn cells were transformed with a plasmid carrying GFP.SmB in combination with the plasmids indicated on the right. Cultures of comparable density were then serially diluted, spotted on EMM2-Leu-Ura- plates in the presence (expression is repressed) or absence (expression is induced) of Thiamine and incubated at 25°C for 5 days to examine their growth ability. The growth defect of either wild-type or tdSMN cells overexpressing Gemin2 is not complemented by an increase in the levels of SmB. tdSMN cells overexpressing both SmB and SpSMN, as expected, grew better than the control. However, the growth of wild-type cells with the same genetic modification was marginally inferior. (C) In GFP.SmB-expressing wild-type cells, SmB is predominantly localised to the nucleus. On upregulation of Gemin2, SmB accumulates in the cytoplasm and cells exhibit an elongated phenotype.
Mentions: Subsequently, we asked whether Gemin2 overexpression is also detrimental in other model organisms, and to this end, we focused on S. pombe, which has been shown to be an excellent system to model human diseases [50]. In particular, we have previously demonstrated that cells carrying a temperature-degron Smn (tdSmn) allele mimic snRNP assembly and splicing defects observed in SMN deficient metazoan cells [34]. Wild-type and tdSmn cells were transformed with a plasmid carrying SpGem2 (yip11) under the control of a very strong nmt1 promoter. Cell cultures of comparable density were subjected to a drop test to investigate their ability to grow at 25°C for 5 days. In case of the tdSmn allele, at a temperature of 25°C, the function of SMN in snRNP assembly is already disrupted [34]. Compared to control (empty plasmid), SpGem2 overexpressors displayed pronounced growth defects in either a wild-type or tdSmn background (Fig 8A). The same cannot be said for SMN. Thus, corroborating our results in Drosophila (above), overexpression of SpSmn had no negative influence on the growth rate of wild-type cells whereas, as expected, it improved growth when overexpressed in a tdSmn background.

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