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Role of gonadotropin regulated testicular RNA helicase (GRTH/Ddx25) on polysomal associated mRNAs in mouse testis.

Tsai-Morris CH, Sato H, Gutti R, Dufau ML - PLoS ONE (2012)

Bottom Line: It is also a negative regulator of steroidogenesis in Leydig cells.A similar analysis was performed using total RNA extracted from purified germ cell populations to address GRTH action in individual target cells.Ingenuity pathway analysis predicted association of GRTH bound polysome genes with the ubiquitin-proteasome-heat shock protein signaling network pathway and NFκB/TP53/TGFB1 signaling networks were derived from the differentially expressed gene analysis.

View Article: PubMed Central - PubMed

Affiliation: Section on Molecular Endocrinology, Program in Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America. morrisch@mail.nih.gov

ABSTRACT
Gonadotropin Regulated Testicular RNA Helicase (GRTH/Ddx25) is a testis-specific multifunctional RNA helicase and an essential post-transcriptional regulator of spermatogenesis. GRTH transports relevant mRNAs from nucleus to cytoplasmic sites of meiotic and haploid germ cells and associates with actively translating polyribosomes. It is also a negative regulator of steroidogenesis in Leydig cells. To obtain a genome-wide perspective of GRTH regulated genes, in particularly those associated with polyribosomes, microarray differential gene expression analysis was performed using polysome-bound RNA isolated from testes of wild type (WT) and GRTH KO mice. 792 genes among the entire mouse genome were found to be polysomal GRTH-linked in WT. Among these 186 were down-regulated and 7 up-regulated genes in GRTH mice. A similar analysis was performed using total RNA extracted from purified germ cell populations to address GRTH action in individual target cells. The down-regulation of known genes concerned with spermatogenesis at polysomal sites in GRTH KO and their association with GRTH in WT coupled with early findings of minor or unchanged total mRNAs and abolition of their protein expression in KO underscore the relevance of GRTH in translation. Ingenuity pathway analysis predicted association of GRTH bound polysome genes with the ubiquitin-proteasome-heat shock protein signaling network pathway and NFκB/TP53/TGFB1 signaling networks were derived from the differentially expressed gene analysis. This study has revealed known and unexplored factors in the genome and regulatory pathways underlying GRTH action in male reproduction.

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Network pathway analysis of mRNA messages associated with GRTH in testicular polysomes of WT mice.(A–E). Five different top score of the associated network pathway predicted by the ingenuity pathway analysis (IPA) are presented. The network was constructed by genes with shape representing functional class of the gene product (www.ingenuity.com). Genes in light pink color are the polysomal mRNA message immunoprecipitated by GRTH antibody. Uncolored genes are predicted by ingenuity pathway knowledge data base as the biological relevance to that network. Solid line: direct interaction. Dotted line: indirect interaction. (F). IPA predicts eight different top score (p<0.5) of molecular and cellular functions in the testicular polysomal mRNA messages associated with GRTH protein in adult mice.
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pone-0032470-g002: Network pathway analysis of mRNA messages associated with GRTH in testicular polysomes of WT mice.(A–E). Five different top score of the associated network pathway predicted by the ingenuity pathway analysis (IPA) are presented. The network was constructed by genes with shape representing functional class of the gene product (www.ingenuity.com). Genes in light pink color are the polysomal mRNA message immunoprecipitated by GRTH antibody. Uncolored genes are predicted by ingenuity pathway knowledge data base as the biological relevance to that network. Solid line: direct interaction. Dotted line: indirect interaction. (F). IPA predicts eight different top score (p<0.5) of molecular and cellular functions in the testicular polysomal mRNA messages associated with GRTH protein in adult mice.

Mentions: To learn the significance of the genes derived from the above microarray analysis in relation to GRTH regulation in the testis, we performed comprehensive bioinformatic analysis using Ingenuity pathway (IPA) software (www.Ingenuity.com) to assess integrated function and pathways of these polysomal GRTH bound mRNAs (792) (Fig. 2) and those GRTH-bound differentially expressed genes revealed in these studies (GRTH WT versus KO) (Fig. 3). An overview of the 792 GRTH-bound mRNAs noted in WT pointed to genes involved in protein synthesis, cellular development, reproductive system development and function among others (Fig. 2F). IPA further reveals five top score networks including genes highly relevant to post-translational modification, protein degradation/synthesis pathway and to cellular function, DNA replication, recombination, tissue development, cellular growth, proliferation and death (Fig. 2A–E). The first network (Fig. 2A) with centered nuclear factor kappa B (NFκB) complex connects to the network of ubiquitin/ubiquitin conjugating enzymes (UBE2:G1, G2, D2, V2, W). This further links to proteasome and ring finger proteins (RNF: 11, 103, 130, 138). Ubiquitin-like modifier activating enzyme 1 (UBA1) is also shown to interact with meiotic synaptonemal complex protein 1 (SYCP1) and histone cluster 1 H2AB/H2AE (Hist1H2AB/HIST1H2AE) (Fig. 2A). The second network (Fig. 2B) includes the heat shock protein complex (HSP) with members, Hsp90 (Hsap90AA1), Hsp70 and Hsp70 like (Hspa1l, Hspa4l), members of Hsp40 family (DNAJB7, DNAJC5B, DNAJA4, DNAJC17, DNAJB4), Hsp27 (HspB1) and Hsp10 (HspE1). Histone cluster 2 H2AC (HIST2H2AC) links to Hsp90 via testis-specific serine kinase 6 (TSSK6). It also interacts with transcriptional regulator, DEK oncogene (DEK). Ddx25 (GRTH) and the germ cells specific messages including outer dense fiber of sperm tails (ODF) and chromatin remodeling transition protein 1 and 2 (TP1/2) are also present in this network. Network 3 includes Akt as centered gene signaling, cAMP dependent protein kinase A (PRKAC) complex and members of A kinase anchor proteins (AKAP). Tubulin associated network with biological function in apoptosis, cell cycle and germ cell-Sertoli cell junction signaling is part of this network (Fig. 2C). Network 4 includes transcriptional coactivator (EP300) and protein kinase C and Prion protein (PRNP) centered pathway (Fig. 2D). Network 5 includes nuclear factor kappa BIA (NFκBIA) (a member of I-κ B proteins which inactives NFκB complex formation) and Dynein (Dnyll1, Dynll2, DnynRB1, DynlT3) centered complexes. DAZ2 restricted to premeiotic spermatogonia is also associated with Dynein associated pathway.


Role of gonadotropin regulated testicular RNA helicase (GRTH/Ddx25) on polysomal associated mRNAs in mouse testis.

Tsai-Morris CH, Sato H, Gutti R, Dufau ML - PLoS ONE (2012)

Network pathway analysis of mRNA messages associated with GRTH in testicular polysomes of WT mice.(A–E). Five different top score of the associated network pathway predicted by the ingenuity pathway analysis (IPA) are presented. The network was constructed by genes with shape representing functional class of the gene product (www.ingenuity.com). Genes in light pink color are the polysomal mRNA message immunoprecipitated by GRTH antibody. Uncolored genes are predicted by ingenuity pathway knowledge data base as the biological relevance to that network. Solid line: direct interaction. Dotted line: indirect interaction. (F). IPA predicts eight different top score (p<0.5) of molecular and cellular functions in the testicular polysomal mRNA messages associated with GRTH protein in adult mice.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316541&req=5

pone-0032470-g002: Network pathway analysis of mRNA messages associated with GRTH in testicular polysomes of WT mice.(A–E). Five different top score of the associated network pathway predicted by the ingenuity pathway analysis (IPA) are presented. The network was constructed by genes with shape representing functional class of the gene product (www.ingenuity.com). Genes in light pink color are the polysomal mRNA message immunoprecipitated by GRTH antibody. Uncolored genes are predicted by ingenuity pathway knowledge data base as the biological relevance to that network. Solid line: direct interaction. Dotted line: indirect interaction. (F). IPA predicts eight different top score (p<0.5) of molecular and cellular functions in the testicular polysomal mRNA messages associated with GRTH protein in adult mice.
Mentions: To learn the significance of the genes derived from the above microarray analysis in relation to GRTH regulation in the testis, we performed comprehensive bioinformatic analysis using Ingenuity pathway (IPA) software (www.Ingenuity.com) to assess integrated function and pathways of these polysomal GRTH bound mRNAs (792) (Fig. 2) and those GRTH-bound differentially expressed genes revealed in these studies (GRTH WT versus KO) (Fig. 3). An overview of the 792 GRTH-bound mRNAs noted in WT pointed to genes involved in protein synthesis, cellular development, reproductive system development and function among others (Fig. 2F). IPA further reveals five top score networks including genes highly relevant to post-translational modification, protein degradation/synthesis pathway and to cellular function, DNA replication, recombination, tissue development, cellular growth, proliferation and death (Fig. 2A–E). The first network (Fig. 2A) with centered nuclear factor kappa B (NFκB) complex connects to the network of ubiquitin/ubiquitin conjugating enzymes (UBE2:G1, G2, D2, V2, W). This further links to proteasome and ring finger proteins (RNF: 11, 103, 130, 138). Ubiquitin-like modifier activating enzyme 1 (UBA1) is also shown to interact with meiotic synaptonemal complex protein 1 (SYCP1) and histone cluster 1 H2AB/H2AE (Hist1H2AB/HIST1H2AE) (Fig. 2A). The second network (Fig. 2B) includes the heat shock protein complex (HSP) with members, Hsp90 (Hsap90AA1), Hsp70 and Hsp70 like (Hspa1l, Hspa4l), members of Hsp40 family (DNAJB7, DNAJC5B, DNAJA4, DNAJC17, DNAJB4), Hsp27 (HspB1) and Hsp10 (HspE1). Histone cluster 2 H2AC (HIST2H2AC) links to Hsp90 via testis-specific serine kinase 6 (TSSK6). It also interacts with transcriptional regulator, DEK oncogene (DEK). Ddx25 (GRTH) and the germ cells specific messages including outer dense fiber of sperm tails (ODF) and chromatin remodeling transition protein 1 and 2 (TP1/2) are also present in this network. Network 3 includes Akt as centered gene signaling, cAMP dependent protein kinase A (PRKAC) complex and members of A kinase anchor proteins (AKAP). Tubulin associated network with biological function in apoptosis, cell cycle and germ cell-Sertoli cell junction signaling is part of this network (Fig. 2C). Network 4 includes transcriptional coactivator (EP300) and protein kinase C and Prion protein (PRNP) centered pathway (Fig. 2D). Network 5 includes nuclear factor kappa BIA (NFκBIA) (a member of I-κ B proteins which inactives NFκB complex formation) and Dynein (Dnyll1, Dynll2, DnynRB1, DynlT3) centered complexes. DAZ2 restricted to premeiotic spermatogonia is also associated with Dynein associated pathway.

Bottom Line: It is also a negative regulator of steroidogenesis in Leydig cells.A similar analysis was performed using total RNA extracted from purified germ cell populations to address GRTH action in individual target cells.Ingenuity pathway analysis predicted association of GRTH bound polysome genes with the ubiquitin-proteasome-heat shock protein signaling network pathway and NFκB/TP53/TGFB1 signaling networks were derived from the differentially expressed gene analysis.

View Article: PubMed Central - PubMed

Affiliation: Section on Molecular Endocrinology, Program in Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America. morrisch@mail.nih.gov

ABSTRACT
Gonadotropin Regulated Testicular RNA Helicase (GRTH/Ddx25) is a testis-specific multifunctional RNA helicase and an essential post-transcriptional regulator of spermatogenesis. GRTH transports relevant mRNAs from nucleus to cytoplasmic sites of meiotic and haploid germ cells and associates with actively translating polyribosomes. It is also a negative regulator of steroidogenesis in Leydig cells. To obtain a genome-wide perspective of GRTH regulated genes, in particularly those associated with polyribosomes, microarray differential gene expression analysis was performed using polysome-bound RNA isolated from testes of wild type (WT) and GRTH KO mice. 792 genes among the entire mouse genome were found to be polysomal GRTH-linked in WT. Among these 186 were down-regulated and 7 up-regulated genes in GRTH mice. A similar analysis was performed using total RNA extracted from purified germ cell populations to address GRTH action in individual target cells. The down-regulation of known genes concerned with spermatogenesis at polysomal sites in GRTH KO and their association with GRTH in WT coupled with early findings of minor or unchanged total mRNAs and abolition of their protein expression in KO underscore the relevance of GRTH in translation. Ingenuity pathway analysis predicted association of GRTH bound polysome genes with the ubiquitin-proteasome-heat shock protein signaling network pathway and NFκB/TP53/TGFB1 signaling networks were derived from the differentially expressed gene analysis. This study has revealed known and unexplored factors in the genome and regulatory pathways underlying GRTH action in male reproduction.

Show MeSH
Related in: MedlinePlus