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.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
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Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
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Moss, Stuart B
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Texas Tech University
Schools of Medicine
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Grozdanov, P N; Li, J; Yu, P et al. (2018) Cstf2t Regulates expression of histones and histone-like proteins in male germ cells. Andrology 6:605-615
MacDonald, Clinton C; Grozdanov, Petar N (2017) Nonsense in the testis: multiple roles for nonsense-mediated decay revealed in male reproduction. Biol Reprod 96:939-947
Harris, Jaryse C; Martinez, Joseph M; Grozdanov, Petar N et al. (2016) The Cstf2t Polyadenylation Gene Plays a Sex-Specific Role in Learning Behaviors in Mice. PLoS One 11:e0165976
Grozdanov, Petar N; Amatullah, Atia; Graber, Joel H et al. (2016) TauCstF-64 Mediates Correct mRNA Polyadenylation and Splicing of Activator and Repressor Isoforms of the Cyclic AMP-Responsive Element Modulator (CREM) in Mouse Testis. Biol Reprod 94:34
Grozdanov, Petar N; MacDonald, Clinton C (2015) Generation of plasmid vectors expressing FLAG-tagged proteins under the regulation of human elongation factor-1? promoter using Gibson assembly. J Vis Exp :
Youngblood, Bradford A; MacDonald, Clinton C (2014) CstF-64 is necessary for endoderm differentiation resulting in cardiomyocyte defects. Stem Cell Res 13:413-21
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
Grozdanov, Petar N; Macdonald, Clinton C (2014) High-throughput sequencing of RNA isolated by cross-linking and immunoprecipitation (HITS-CLIP) to determine sites of binding of CstF-64 on nascent RNAs. Methods Mol Biol 1125:187-208
Youngblood, Bradford A; Grozdanov, Petar N; MacDonald, Clinton C (2014) CstF-64 supports pluripotency and regulates cell cycle progression in embryonic stem cells through histone 3' end processing. Nucleic Acids Res 42:8330-42
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

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