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Conserved genes act as modifiers of invertebrate SMN loss of function defects.

Dimitriadi M, Sleigh JN, Walker A, Chang HC, Sen A, Kalloo G, Harris J, Barsby T, Walsh MB, Satterlee JS, Li C, Van Vactor D, Artavanis-Tsakonas S, Hart AC - PLoS Genet. (2010)

Bottom Line: Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model.Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects.Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Brown University, Providence, Rhode Island, USA.

ABSTRACT
Spinal Muscular Atrophy (SMA) is caused by diminished function of the Survival of Motor Neuron (SMN) protein, but the molecular pathways critical for SMA pathology remain elusive. We have used genetic approaches in invertebrate models to identify conserved SMN loss of function modifier genes. Drosophila melanogaster and Caenorhabditis elegans each have a single gene encoding a protein orthologous to human SMN; diminished function of these invertebrate genes causes lethality and neuromuscular defects. To find genes that modulate SMN function defects across species, two approaches were used. First, a genome-wide RNAi screen for C. elegans SMN modifier genes was undertaken, yielding four genes. Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model. Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects. C. elegans orthologs of twelve genes, which were originally identified in a previous Drosophila screen, modified C. elegans SMN loss of function defects. Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species.

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Genome-wide RNAi screen for Cesmn-1(lf) modifier genes.A) To identify genes whose knockdown modifies the growth of Cesmn-1(lf) animals, the progeny of hT2(lethal)[myo-2p::GFP]/Cesmn-1(lf) (+/Cesmn-1(lf), described in text) were reared for more than 2 generations in 96-well, liquid culture format on bacteria expressing dsRNA corresponding to over 16,500 C. elegans genes. Modifier genes identified in this assay were tested subsequently in neuromuscular assays in C. elegans and Drosophila. B) Cesmn-1(lf) and +/Cesmn-1(lf) length was measured for each RNAi clone and a ‘growth’ ratio of large∶small animals was determined for each genotype. Representative graphs illustrate the distribution of RNAi clone growth ratios. Candidate enhancer genes were those with a growth ratio more than 2 standard deviations above the mean for Cesmn-1(lf) (shaded in right graph) and within 0.7 standard deviations of the mean for +/Cesmn-1(lf) (shaded in left graph) for each 96-well plate. No suppressors were identified using similar criteria. Two independent determinations were made for each clone in the original screen. Candidate genes were retested in at least quadruplicate and enhanced growth in at least 40% of trials before designation as growth modifier genes; average growth ratios of enhancers from the C. elegans screen are indicated.
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pgen-1001172-g002: Genome-wide RNAi screen for Cesmn-1(lf) modifier genes.A) To identify genes whose knockdown modifies the growth of Cesmn-1(lf) animals, the progeny of hT2(lethal)[myo-2p::GFP]/Cesmn-1(lf) (+/Cesmn-1(lf), described in text) were reared for more than 2 generations in 96-well, liquid culture format on bacteria expressing dsRNA corresponding to over 16,500 C. elegans genes. Modifier genes identified in this assay were tested subsequently in neuromuscular assays in C. elegans and Drosophila. B) Cesmn-1(lf) and +/Cesmn-1(lf) length was measured for each RNAi clone and a ‘growth’ ratio of large∶small animals was determined for each genotype. Representative graphs illustrate the distribution of RNAi clone growth ratios. Candidate enhancer genes were those with a growth ratio more than 2 standard deviations above the mean for Cesmn-1(lf) (shaded in right graph) and within 0.7 standard deviations of the mean for +/Cesmn-1(lf) (shaded in left graph) for each 96-well plate. No suppressors were identified using similar criteria. Two independent determinations were made for each clone in the original screen. Candidate genes were retested in at least quadruplicate and enhanced growth in at least 40% of trials before designation as growth modifier genes; average growth ratios of enhancers from the C. elegans screen are indicated.

Mentions: To identify additional genes that modify SMN loss of function defects, a large-scale genome-wide screen for enhancers and suppressors of the Cesmn-1(lf) growth defect was undertaken. The growth assay was adapted to a higher-throughput 96-well, liquid culture format and a previously described genome-wide C. elegans RNAi feeding library was used for gene knockdown (Figure 2A) [53]. Progeny of +/Cesmn-1(lf) animals were reared for two weeks (more than 2 generations) on RNAi feeding bacterial strains before assessment of growth using the COPAS Biosorter [51]. To identify RNAi clones that specifically altered the growth of Cesmn-1(lf) animals, a growth ratio of large to small animals was determined for each clone for Cesmn-1(lf) and for +/Cesmn-1(lf) genotypes. If the RNAi clone growth ratio was more than 2 standard deviations away from the mean for Cesmn-1(lf) animals and within 0.7 standard deviations of the mean for +/Cesmn-1(lf) animals in at least 40% of independent trials, then the corresponding gene was designated as an Cesmn-1(lf) modifier (Figure 2B). In the primary high-throughput screen, no suppressors were found, but four genes were identified as enhancers (Figure 2B). RNAi knockdown of these genes exacerbated homozygous Cesmn-1(lf) growth defects and did not significantly alter the growth of heterozygous +/Cesmn-1(lf) animals: ncbp-2, T02G5.3, grk-2, and flp-4. ncbp-2 encodes the C. elegans Cap Binding Protein 20 (CBP20 or Cbp20) ortholog [54]. T02G5.3 encodes a predicted protein of unknown function with no vertebrate orthologs based on BLAST analysis. grk-2 encodes one of two G-protein coupled receptor kinases. flp-4 encodes an FMRFamide family neuropeptide protein. The low number of modifiers identified in this screen versus the previous Drosophila screen may reflect the stringent criterion utilized here or the inefficiency of RNAi by feeding in neurons.


Conserved genes act as modifiers of invertebrate SMN loss of function defects.

Dimitriadi M, Sleigh JN, Walker A, Chang HC, Sen A, Kalloo G, Harris J, Barsby T, Walsh MB, Satterlee JS, Li C, Van Vactor D, Artavanis-Tsakonas S, Hart AC - PLoS Genet. (2010)

Genome-wide RNAi screen for Cesmn-1(lf) modifier genes.A) To identify genes whose knockdown modifies the growth of Cesmn-1(lf) animals, the progeny of hT2(lethal)[myo-2p::GFP]/Cesmn-1(lf) (+/Cesmn-1(lf), described in text) were reared for more than 2 generations in 96-well, liquid culture format on bacteria expressing dsRNA corresponding to over 16,500 C. elegans genes. Modifier genes identified in this assay were tested subsequently in neuromuscular assays in C. elegans and Drosophila. B) Cesmn-1(lf) and +/Cesmn-1(lf) length was measured for each RNAi clone and a ‘growth’ ratio of large∶small animals was determined for each genotype. Representative graphs illustrate the distribution of RNAi clone growth ratios. Candidate enhancer genes were those with a growth ratio more than 2 standard deviations above the mean for Cesmn-1(lf) (shaded in right graph) and within 0.7 standard deviations of the mean for +/Cesmn-1(lf) (shaded in left graph) for each 96-well plate. No suppressors were identified using similar criteria. Two independent determinations were made for each clone in the original screen. Candidate genes were retested in at least quadruplicate and enhanced growth in at least 40% of trials before designation as growth modifier genes; average growth ratios of enhancers from the C. elegans screen are indicated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965752&req=5

pgen-1001172-g002: Genome-wide RNAi screen for Cesmn-1(lf) modifier genes.A) To identify genes whose knockdown modifies the growth of Cesmn-1(lf) animals, the progeny of hT2(lethal)[myo-2p::GFP]/Cesmn-1(lf) (+/Cesmn-1(lf), described in text) were reared for more than 2 generations in 96-well, liquid culture format on bacteria expressing dsRNA corresponding to over 16,500 C. elegans genes. Modifier genes identified in this assay were tested subsequently in neuromuscular assays in C. elegans and Drosophila. B) Cesmn-1(lf) and +/Cesmn-1(lf) length was measured for each RNAi clone and a ‘growth’ ratio of large∶small animals was determined for each genotype. Representative graphs illustrate the distribution of RNAi clone growth ratios. Candidate enhancer genes were those with a growth ratio more than 2 standard deviations above the mean for Cesmn-1(lf) (shaded in right graph) and within 0.7 standard deviations of the mean for +/Cesmn-1(lf) (shaded in left graph) for each 96-well plate. No suppressors were identified using similar criteria. Two independent determinations were made for each clone in the original screen. Candidate genes were retested in at least quadruplicate and enhanced growth in at least 40% of trials before designation as growth modifier genes; average growth ratios of enhancers from the C. elegans screen are indicated.
Mentions: To identify additional genes that modify SMN loss of function defects, a large-scale genome-wide screen for enhancers and suppressors of the Cesmn-1(lf) growth defect was undertaken. The growth assay was adapted to a higher-throughput 96-well, liquid culture format and a previously described genome-wide C. elegans RNAi feeding library was used for gene knockdown (Figure 2A) [53]. Progeny of +/Cesmn-1(lf) animals were reared for two weeks (more than 2 generations) on RNAi feeding bacterial strains before assessment of growth using the COPAS Biosorter [51]. To identify RNAi clones that specifically altered the growth of Cesmn-1(lf) animals, a growth ratio of large to small animals was determined for each clone for Cesmn-1(lf) and for +/Cesmn-1(lf) genotypes. If the RNAi clone growth ratio was more than 2 standard deviations away from the mean for Cesmn-1(lf) animals and within 0.7 standard deviations of the mean for +/Cesmn-1(lf) animals in at least 40% of independent trials, then the corresponding gene was designated as an Cesmn-1(lf) modifier (Figure 2B). In the primary high-throughput screen, no suppressors were found, but four genes were identified as enhancers (Figure 2B). RNAi knockdown of these genes exacerbated homozygous Cesmn-1(lf) growth defects and did not significantly alter the growth of heterozygous +/Cesmn-1(lf) animals: ncbp-2, T02G5.3, grk-2, and flp-4. ncbp-2 encodes the C. elegans Cap Binding Protein 20 (CBP20 or Cbp20) ortholog [54]. T02G5.3 encodes a predicted protein of unknown function with no vertebrate orthologs based on BLAST analysis. grk-2 encodes one of two G-protein coupled receptor kinases. flp-4 encodes an FMRFamide family neuropeptide protein. The low number of modifiers identified in this screen versus the previous Drosophila screen may reflect the stringent criterion utilized here or the inefficiency of RNAi by feeding in neurons.

Bottom Line: Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model.Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects.Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Brown University, Providence, Rhode Island, USA.

ABSTRACT
Spinal Muscular Atrophy (SMA) is caused by diminished function of the Survival of Motor Neuron (SMN) protein, but the molecular pathways critical for SMA pathology remain elusive. We have used genetic approaches in invertebrate models to identify conserved SMN loss of function modifier genes. Drosophila melanogaster and Caenorhabditis elegans each have a single gene encoding a protein orthologous to human SMN; diminished function of these invertebrate genes causes lethality and neuromuscular defects. To find genes that modulate SMN function defects across species, two approaches were used. First, a genome-wide RNAi screen for C. elegans SMN modifier genes was undertaken, yielding four genes. Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model. Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects. C. elegans orthologs of twelve genes, which were originally identified in a previous Drosophila screen, modified C. elegans SMN loss of function defects. Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species.

Show MeSH
Related in: MedlinePlus