Murray State University

Reverse Genetic Analysis Indicates Redundancy in the M1 Aminopeptidase Family of Caenorhabditis elegans

Institution

Murray State University

Abstract

Mutations of the puromycin sensitive aminopeptidase (Psa) orthologs of flies, mice, and plants result in meiotic errors and reduced embryonic viability. Genetic lesions of the Caenorhabditis elegans ortholog of Psa, pam-1, similarly result in a dramatic reduction of worm fecundity. PAM-1, an M1 aminopeptidase, normally functions in the somatic gonad tissue where it activates the conserved germinal LET-60/Ras, MEK-2/Mek1, and MPK-1/Erk1 signaling axis which ultimately triggers the exit from pachytene and nucleolar breakdown during oogenesis. Worms harboring mutant pam-1(ne4176) and pam-1 (or282) alleles have a compromised signaling pathway which is manifested as an expanded population of pachytene germ cell nuclei toward the proximal end of the gonad and a delayed nucleolar breakdown, ultimately hindering embryonic viability and overall brood sizes. Based on the homology between the amino acid sequences of eight additional M1 aminopeptidase proteins encoded within the C. elegans genome, sequence analysis suggests that one or more of these proteins may also function in regulating C. elegans fertility. Using RNAi techniques, we have systematically evaluated the effects of inactivating the expression of the functional aminopeptidase paralogs individually in the wild type (N2) worm or in combination with the reduced-function pam-1(ne4176) allele and loss-of-function pam-1(or282) allele. We demonstrate compensatory paralog function affecting worm fertility with observed statistically significant decreases among both overall brood sizes and embryonic viability. Evidence of exacerbation within the germ cell phenotype of pachytene extension and delayed nucleolar breakdown compliments the fecundity data, suggesting that multiple M1 aminopeptidases have compensatory functions in the C. elegans gonad.

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Reverse Genetic Analysis Indicates Redundancy in the M1 Aminopeptidase Family of Caenorhabditis elegans

Mutations of the puromycin sensitive aminopeptidase (Psa) orthologs of flies, mice, and plants result in meiotic errors and reduced embryonic viability. Genetic lesions of the Caenorhabditis elegans ortholog of Psa, pam-1, similarly result in a dramatic reduction of worm fecundity. PAM-1, an M1 aminopeptidase, normally functions in the somatic gonad tissue where it activates the conserved germinal LET-60/Ras, MEK-2/Mek1, and MPK-1/Erk1 signaling axis which ultimately triggers the exit from pachytene and nucleolar breakdown during oogenesis. Worms harboring mutant pam-1(ne4176) and pam-1 (or282) alleles have a compromised signaling pathway which is manifested as an expanded population of pachytene germ cell nuclei toward the proximal end of the gonad and a delayed nucleolar breakdown, ultimately hindering embryonic viability and overall brood sizes. Based on the homology between the amino acid sequences of eight additional M1 aminopeptidase proteins encoded within the C. elegans genome, sequence analysis suggests that one or more of these proteins may also function in regulating C. elegans fertility. Using RNAi techniques, we have systematically evaluated the effects of inactivating the expression of the functional aminopeptidase paralogs individually in the wild type (N2) worm or in combination with the reduced-function pam-1(ne4176) allele and loss-of-function pam-1(or282) allele. We demonstrate compensatory paralog function affecting worm fertility with observed statistically significant decreases among both overall brood sizes and embryonic viability. Evidence of exacerbation within the germ cell phenotype of pachytene extension and delayed nucleolar breakdown compliments the fecundity data, suggesting that multiple M1 aminopeptidases have compensatory functions in the C. elegans gonad.