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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD037109-11
Application #
8422877
Study Section
Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
Program Officer
Moss, Stuart B
Project Start
1999-09-27
Project End
2016-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
11
Fiscal Year
2013
Total Cost
$298,468
Indirect Cost
$96,805
Name
Texas Tech University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
609980727
City
Lubbock
State
TX
Country
United States
Zip Code
79430
Alfano, Randall; Youngblood, Bradford A; Zhang, Deshui et al. (2014) Human leukemia inhibitory factor produced by the ExpressTec method from rice (Oryza sativa L.) is active in human neural stem cells and mouse induced pluripotent stem cells. Bioengineered 5:180-5
Youngblood, Bradford A; MacDonald, Clinton C (2014) CstF-64 is necessary for endoderm differentiation resulting in cardiomyocyte defects. Stem Cell Res 13:413-21
Youngblood, Bradford A; Alfano, Randall; Pettit, Steve C et al. (2014) Application of recombinant human leukemia inhibitory factor (LIF) produced in rice (Oryza sativa L.) for maintenance of mouse embryonic stem cells. J Biotechnol 172:67-72
Shankarling, Ganesh S; MacDonald, Clinton C (2013) Polyadenylation site-specific differences in the activity of the neuronal *CstF-64 protein in PC-12 cells. Gene 529:220-7
Hockert, J Andrew; Yeh, Hsiang-Jui; MacDonald, Clinton C (2010) The hinge domain of the cleavage stimulation factor protein CstF-64 is essential for CstF-77 interaction, nuclear localization, and polyadenylation. J Biol Chem 285:695-704
Shankarling, Ganesh S; Coates, Penelope W; Dass, Brinda et al. (2009) A family of splice variants of CstF-64 expressed in vertebrate nervous systems. BMC Mol Biol 10:22
Dass, Brinda; Tardif, Steve; Park, Ji Yeon et al. (2007) Loss of polyadenylation protein tauCstF-64 causes spermatogenic defects and male infertility. Proc Natl Acad Sci U S A 104:20374-9
Monarez, Roberto R; MacDonald, Clinton C; Dass, Brinda (2007) Polyadenylation proteins CstF-64 and tauCstF-64 exhibit differential binding affinities for RNA polymers. Biochem J 401:651-8
D'mello, Veera; Lee, Ju Y; MacDonald, Clinton C et al. (2006) Alternative mRNA polyadenylation can potentially affect detection of gene expression by affymetrix genechip arrays. Appl Bioinformatics 5:249-53
McMahon, K Wyatt; Hirsch, Benjamin A; MacDonald, Clinton C (2006) Differences in polyadenylation site choice between somatic and male germ cells. BMC Mol Biol 7:35

Showing the most recent 10 out of 15 publications