We are trying to understand how mRNA polyadenylation occurs in male germ cells in the absence of the canonical AAUAAA polyadenylation signal. During our previous grant period we discovered and characterized a variant form of the CstF-64 polyadenylation protein that is expressed at high levels in meiotic and postmeiotic germ cells, and in no other tissue except the brain. We cloned and sequenced this new protein, called """"""""tauCstF-64,"""""""" and it is the leading candidate to account for AAUAAA-independent polyadenylation in germ cells. Our overall laboratory hypothesis is that (1) the somatic CstF-64 gene (Cstf2) is on the X chromosome in mice and humans, (2) the Xchromosomal CstF-64 is inactivated during male meiosis, (3) since CstF-64 function is essential, an auxiliary CstF-64 gene must be expressed from an autosome, and (4) the auxiliary CstF-64 protein is responsible for AAUAAA-independent polyadenylation of mRNAs in male germ cells and is essential for spermatogenesis. In the previous grant period we addressed items 1-3 of our laboratory hypothesis. In this application we propose to address item 4 or our laboratory hypothesis: we will determine whether specific domains of tauCstF-64 contribute to germ cell polyadenylation. We will also determine whether tauCstF-64 is sufficient for AAUAAA-independent polyadenylation by expressing it ectopically in cells in culture, and whether it is necessary for spermatogenesis by creating a transgenic strain of mice that lack the Cstf2t gene for tauCstF-64. Finally, we will begin to examine tauCstF-64 function in human germ cells by examining its distribution in human testis and other tissues.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
3R01HD037109-05S1
Application #
6871948
Study Section
Reproductive Endocrinology Study Section (REN)
Program Officer
Taymans, Susan
Project Start
1998-12-01
Project End
2007-05-31
Budget Start
2003-06-01
Budget End
2004-05-31
Support Year
5
Fiscal Year
2004
Total Cost
$17,300
Indirect Cost
Name
Texas Tech University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
609980727
City
Lubbock
State
TX
Country
United States
Zip Code
79430
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 :
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
Hockert, J Andrew; Macdonald, Clinton C (2014) The stem-loop luciferase assay for polyadenylation (SLAP) method for determining CstF-64-dependent polyadenylation activity. Methods Mol Biol 1125:109-17

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