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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

Rescue of lethality associated with homozygous and transheterozygous Gemin5 allelic combinations.(A) Genomic and genetic contexts of the Gemin5 gene locus. The CG13436 gene is nested within intron 4 of the Gemin5 gene. Locations of two P-element insertions in the 5’ UTR, Gem5P and Gem5W, as well as, Gem5M, a PiggyBac insertion in exon 2 are indicated. (B) Analysis of the percentage number (mean ± S.E.M. of at least 4 independent experiments) of viable adults with ubiquitous transgenic expression of Gemin5 and the respective allelic combinations. Numbers of flies analysed are indicated in parenthesis. Gem5M homozygotes and transheterozygous mutant combinations all show adequate level of rescue. Rescue is absent and sub-optimal for Gem5P and Gem5W homozygotes, respectively.
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pone.0130974.g001: Rescue of lethality associated with homozygous and transheterozygous Gemin5 allelic combinations.(A) Genomic and genetic contexts of the Gemin5 gene locus. The CG13436 gene is nested within intron 4 of the Gemin5 gene. Locations of two P-element insertions in the 5’ UTR, Gem5P and Gem5W, as well as, Gem5M, a PiggyBac insertion in exon 2 are indicated. (B) Analysis of the percentage number (mean ± S.E.M. of at least 4 independent experiments) of viable adults with ubiquitous transgenic expression of Gemin5 and the respective allelic combinations. Numbers of flies analysed are indicated in parenthesis. Gem5M homozygotes and transheterozygous mutant combinations all show adequate level of rescue. Rescue is absent and sub-optimal for Gem5P and Gem5W homozygotes, respectively.

Mentions: The Drosophila Gemin5 gene consists of 9 exons that encode for an approximately 138 kDa protein with a high conservation to its human counterpart especially within the WD repeat domain-rich N-terminus [37]. Gates et al. [43] reported that Gemin5 transposon insertion mutants died in their majority as third instar larvae prior to developing moulting defects. However, the presence of a nested gene (CG13436) within intron 4 (Fig 1A), which might also be disrupted, raises questions about the specificity of the Gemin5 mutants. Attempting at clarifying this issue as well as confirming the functionality of a Gemin5 transgene, we performed rescue analysis on homozygous and transheterozygous allelic combinations. Via complementation crosses we first confirmed that the two mutants described by Gates et al. [43], including Gem5P and Gem5W each having a P-element insert in the 5’ UTR, retain their recessive lethality in trans to each other (Gem5W/Gem5P) and to chromosomal deficiencies that completely abolish the Gemin5 gene amongst others (Gem5W/Df(2R)exu1, Gem5W/Df(2R)exu2, Gem5P/Df(2R)exu1, and Gem5W/Df(2R)exu2). In addition, Gem5M, a new mutant with a PiggyBac insertion in the 2nd exon was also lethal in the homozygous state (Gem5M/Gem5M) or when combined with Gem5P (Gem5M/Gem5P), Gem5W (Gem5M/Gem5W) or chromosomal deficiencies (Gem5M/Df(2R)exu1, and Gem5M/Df(2R)exu2) in a transheterozygous state. Ubiquitous expression of a Gemin5 transgene driven by da-GAL4 rescued the lethality of both Gem5M and Gem5W homozygotes, although the degree of rescue was higher in the former compared to the latter (Fig 1B). No rescue was obtained for Gem5P homozygotes (Gem5P/Gem5P) exposing the influence of a non-specific mutation on its lethal phenotype. Transheterozygous combinations of all three alleles, including Gem5M/Gem5P, Gem5M/Gem5W, and Gem5P/Gem5W, were rescued to a similar degree, with the level of rescue being similar or higher to that of Gem5M homozygotes. These findings attest to the functionality of the Gemin5 transgene utilised, hence, allowing its use in downstream experiments.


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

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

Rescue of lethality associated with homozygous and transheterozygous Gemin5 allelic combinations.(A) Genomic and genetic contexts of the Gemin5 gene locus. The CG13436 gene is nested within intron 4 of the Gemin5 gene. Locations of two P-element insertions in the 5’ UTR, Gem5P and Gem5W, as well as, Gem5M, a PiggyBac insertion in exon 2 are indicated. (B) Analysis of the percentage number (mean ± S.E.M. of at least 4 independent experiments) of viable adults with ubiquitous transgenic expression of Gemin5 and the respective allelic combinations. Numbers of flies analysed are indicated in parenthesis. Gem5M homozygotes and transheterozygous mutant combinations all show adequate level of rescue. Rescue is absent and sub-optimal for Gem5P and Gem5W homozygotes, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130974.g001: Rescue of lethality associated with homozygous and transheterozygous Gemin5 allelic combinations.(A) Genomic and genetic contexts of the Gemin5 gene locus. The CG13436 gene is nested within intron 4 of the Gemin5 gene. Locations of two P-element insertions in the 5’ UTR, Gem5P and Gem5W, as well as, Gem5M, a PiggyBac insertion in exon 2 are indicated. (B) Analysis of the percentage number (mean ± S.E.M. of at least 4 independent experiments) of viable adults with ubiquitous transgenic expression of Gemin5 and the respective allelic combinations. Numbers of flies analysed are indicated in parenthesis. Gem5M homozygotes and transheterozygous mutant combinations all show adequate level of rescue. Rescue is absent and sub-optimal for Gem5P and Gem5W homozygotes, respectively.
Mentions: The Drosophila Gemin5 gene consists of 9 exons that encode for an approximately 138 kDa protein with a high conservation to its human counterpart especially within the WD repeat domain-rich N-terminus [37]. Gates et al. [43] reported that Gemin5 transposon insertion mutants died in their majority as third instar larvae prior to developing moulting defects. However, the presence of a nested gene (CG13436) within intron 4 (Fig 1A), which might also be disrupted, raises questions about the specificity of the Gemin5 mutants. Attempting at clarifying this issue as well as confirming the functionality of a Gemin5 transgene, we performed rescue analysis on homozygous and transheterozygous allelic combinations. Via complementation crosses we first confirmed that the two mutants described by Gates et al. [43], including Gem5P and Gem5W each having a P-element insert in the 5’ UTR, retain their recessive lethality in trans to each other (Gem5W/Gem5P) and to chromosomal deficiencies that completely abolish the Gemin5 gene amongst others (Gem5W/Df(2R)exu1, Gem5W/Df(2R)exu2, Gem5P/Df(2R)exu1, and Gem5W/Df(2R)exu2). In addition, Gem5M, a new mutant with a PiggyBac insertion in the 2nd exon was also lethal in the homozygous state (Gem5M/Gem5M) or when combined with Gem5P (Gem5M/Gem5P), Gem5W (Gem5M/Gem5W) or chromosomal deficiencies (Gem5M/Df(2R)exu1, and Gem5M/Df(2R)exu2) in a transheterozygous state. Ubiquitous expression of a Gemin5 transgene driven by da-GAL4 rescued the lethality of both Gem5M and Gem5W homozygotes, although the degree of rescue was higher in the former compared to the latter (Fig 1B). No rescue was obtained for Gem5P homozygotes (Gem5P/Gem5P) exposing the influence of a non-specific mutation on its lethal phenotype. Transheterozygous combinations of all three alleles, including Gem5M/Gem5P, Gem5M/Gem5W, and Gem5P/Gem5W, were rescued to a similar degree, with the level of rescue being similar or higher to that of Gem5M homozygotes. These findings attest to the functionality of the Gemin5 transgene utilised, hence, allowing its use in downstream experiments.

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