MRNA PROCESSING IN REPRODUCTION AND FERTILIZATION

Summary

Principal Investigator: Clinton C MacDonald
Abstract: DESCRIPTION (provided by applicant): Polyadenylation-the addition of a poly(A) tail to an mRNA 32 end-is essential for gene expression in every tissue. However, fundamental features of polyadenylation-the RNA signals used, the sites chosen, and the proteins involved-are different in male germ cells than in any other tissue. These differences are essential for the correct expression of key genes during spermatogenesis, for example altering the protein forms of transcription factors or altering translational efficiency. DCstF-64 (gene name: Cstf2t) is the testis-expressed variant of CstF-64, an RNA-binding protein that regulates polyadenylation in somatic cells. DCstF-64 is essential for normal spermatogenesis: male Cstf2t knockout mice are infertile due to severe defects in meiotic and postmeiotic sperm production, a condition that resembles oligoasthenoteratozoospermia in human patients. We have found that Cstf2t controls polyadenylation of at least two important classes of genes, long interspersed nuclear elements (LINEs) and intronless small genes (ISGs). LINEs are mobile elements that are responsible for at least 70 genetic diseases, thus being a genomic and metabolic burden. DCstF-64 reduces LINE mRNAs by promoting polyadenylation at internal sites in the gene, thus reducing their abundance and suppressing their proliferation. To determine how DCstF-64 controls LINE expression, we will test whether exogenous DCstF-64 will suppress LINE mRNA expression, whether DCstF-64 binds to LINE mRNAs at internal polyadenylation sites, and whether exogenous DCstF-64 will suppress rates of retrotransposition in a cell culture assay. ISGs are expressed retroposons that control major functions in spermatogenesis (metabolism, gene expression, chromosome structure, and more). To determine how DCstF-64 regulates polyadenylation and termination of ISG mRNAs, we will test whether DCstF-64 is associated preferentially with ISG polyadenylation sites, whether exogenous DCstF-64 is required for correct polyadenylation of ISGs, and whether DCstF-64 is required for normal transcriptional termination of ISGs. Finally, to determine germ cell-specific functions of DCstF-64, we will perform a genetic test to determine whether CstF-64 will complement the Cstf2ttm1Ccma infertility phenotype, purify DCstF-64 complexes to look for germ cell-specific components, and test functions of DCstF-64 domains using a luciferase-based cell transfection assay and transgenic mice. PUBLIC HEALTH RELEVANCE: The most heartbreaking of disorders can be infertility, when a couple fails to conceive after at least a year of attempts. We discovered a gene, CSTF2T, that can be a hidden cause of male infertility-hidden, because females missing this gene have normal fertility, while males have severe problems in sperm production. Using a variety of techniques, we want to learn the molecular causes of why sperm production fails in males missing the CSTF2T gene, to better understand how to assist these infertile couples.
Funding Period: 1999-09-27 - 2016-01-31
more information: NIH RePORT

Top Publications

  1. pmc Polyadenylation proteins CstF-64 and tauCstF-64 exhibit differential binding affinities for RNA polymers
    Roberto R Monarez
    Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430, USA
    Biochem J 401:651-8. 2007
  2. pmc Differences in polyadenylation site choice between somatic and male germ cells
    K Wyatt McMahon
    Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430 6540 USA
    BMC Mol Biol 7:35. 2006
  3. ncbi Alternative mRNA polyadenylation can potentially affect detection of gene expression by affymetrix genechip arrays
    Veera D'mello
    Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07101, USA
    Appl Bioinformatics 5:249-53. 2006
  4. pmc Loss of polyadenylation protein tauCstF-64 causes spermatogenic defects and male infertility
    Brinda Dass
    Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
    Proc Natl Acad Sci U S A 104:20374-9. 2007
  5. pmc A family of splice variants of CstF-64 expressed in vertebrate nervous systems
    Ganesh S Shankarling
    Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430 6540, USA
    BMC Mol Biol 10:22. 2009
  6. pmc The hinge domain of the cleavage stimulation factor protein CstF-64 is essential for CstF-77 interaction, nuclear localization, and polyadenylation
    J Andrew Hockert
    Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430 6540, USA
    J Biol Chem 285:695-704. 2010
  7. pmc Spermatogenetic but not immunological defects in mice lacking the τCstF-64 polyadenylation protein
    Kathy Jo Hockert
    Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Texas, Lubbock, TX 79430, USA
    J Reprod Immunol 89:26-37. 2011
  8. pmc Polyadenylation site-specific differences in the activity of the neuronal βCstF-64 protein in PC-12 cells
    Ganesh S Shankarling
    Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430 6540, USA
    Gene 529:220-7. 2013
  9. pmc Application of recombinant human leukemia inhibitory factor (LIF) produced in rice (Oryza sativa L.) for maintenance of mouse embryonic stem cells
    Bradford A Youngblood
    Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430 6540, USA
    J Biotechnol 172:67-72. 2014

Detail Information

Publications9

  1. pmc Polyadenylation proteins CstF-64 and tauCstF-64 exhibit differential binding affinities for RNA polymers
    Roberto R Monarez
    Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430, USA
    Biochem J 401:651-8. 2007
    ..This supports the hypothesis that tauCstF-64 promotes germ-cell-specific patterns of polyadenylation by binding to different downstream sequence elements...
  2. pmc Differences in polyadenylation site choice between somatic and male germ cells
    K Wyatt McMahon
    Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430 6540 USA
    BMC Mol Biol 7:35. 2006
    ..We therefore hypothesized that male germ cell-specific polyadenylation sites would be inefficiently used in somatic cells...
  3. ncbi Alternative mRNA polyadenylation can potentially affect detection of gene expression by affymetrix genechip arrays
    Veera D'mello
    Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07101, USA
    Appl Bioinformatics 5:249-53. 2006
    ..This could lead to inaccurate interpretation of GeneChip data when the changes of expression values actually result from alternative use of polyadenylation sites...
  4. pmc Loss of polyadenylation protein tauCstF-64 causes spermatogenic defects and male infertility
    Brinda Dass
    Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
    Proc Natl Acad Sci U S A 104:20374-9. 2007
    ..Finally, these transgenic mice provide a model for the study of polyadenylation in an isolated in vivo system and highlight the role of a growing family of testis-expressed autosomal retroposed variants of X-linked genes...
  5. pmc A family of splice variants of CstF-64 expressed in vertebrate nervous systems
    Ganesh S Shankarling
    Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430 6540, USA
    BMC Mol Biol 10:22. 2009
    ..Since the CstF-64 polyadenylation protein is known to be an important regulator of tissue-specific polyadenylation, we examined its expression in brain and other organs...
  6. pmc The hinge domain of the cleavage stimulation factor protein CstF-64 is essential for CstF-77 interaction, nuclear localization, and polyadenylation
    J Andrew Hockert
    Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430 6540, USA
    J Biol Chem 285:695-704. 2010
    ..Further, we showed that the Hinge domain is necessary for CstF-64 interaction with CstF-77 and consequent nuclear localization, suggesting that nuclear import of a preformed CstF complex is an essential step in polyadenylation...
  7. pmc Spermatogenetic but not immunological defects in mice lacking the τCstF-64 polyadenylation protein
    Kathy Jo Hockert
    Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Texas, Lubbock, TX 79430, USA
    J Reprod Immunol 89:26-37. 2011
    ..Together, these data show that τCstF-64 function is primarily to support spermatogenesis, but only incidentally to support immune cell function...
  8. pmc Polyadenylation site-specific differences in the activity of the neuronal βCstF-64 protein in PC-12 cells
    Ganesh S Shankarling
    Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430 6540, USA
    Gene 529:220-7. 2013
    ..Our data address the polyadenylation functions of βCstF-64 for the first time, and provide initial insights into the mechanism of alternative poly(A) site selection in the nervous system. ..
  9. pmc Application of recombinant human leukemia inhibitory factor (LIF) produced in rice (Oryza sativa L.) for maintenance of mouse embryonic stem cells
    Bradford A Youngblood
    Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430 6540, USA
    J Biotechnol 172:67-72. 2014
    ..Retention of the pluripotent state was found to be indistinguishable between rice-derived rhLIF and other recombinant LIF proteins currently on the market. ..