Both male and female germ cells are confron-ted with the need to stabilize mRNAs. This application proposes that the germ cell-specific Y-box RNA-binding protein, MSY2, is essential to maintain global mRNA stability in male and female germ cells. The high level of MSY2 protein in meiotic and in post-meiotic male germ cells, coupled with its apparent sequence-independent RNA-binding activity and ability to suppress translation, supports this hypothesis. Likewise, in the growing oocyte, the very high abundance of MSY2 protein and its destruction by the 2-cell stage, coincident with the degradation of maternal mRNAs, supports this hypothesis.
Specific Aim 1 will test the hypothesis that MSY2 is a global regulator of mRNA stability in male germ cells and oocytes by (1) reducing the amount of MSY2 protein by two complementary approaches, i.e., conventional knockout and transgenic RNAi, and (2) increasing MSY2 protein levels in male germ cells and oocytes by transgenesis. The coordinate degradation of MSY2 and maternal mRNAs suggests a linkage between MSY2 turnover and mRNA destruction. The turnover of maternal mRNA is essential for continued embryo development, suggesting MSY2 degradation is a crucial regulatory event. There is a growing body of evidence that one function of protein phosphorylation is to mark the phosphorylated protein for degradation.
Specific Aim 2 will test the hypothesis that specific maturation-associated phosphorylations of MSY2 in oocytes mark MSY2 for degradation that is mechanistically linked to the degradation of maternal mRNA by (1) determining the sites of phosphorylation, and (2) assessing the effect of mutating these sites on both MSY2 protein and mRNA degradation and the functional consequences on preimplantation development. The high levels of protein synthesis in meiotic and postmeiotic male germ cells necessitates the continued translation of many mRNAs, which raises the question how this occurs in the presence of a high level of MSY2.
Specific Aim 3 will test the hypothesis that, although the majority of male germ cell mRNAs are associated with MSY2, a subpopulation not under translational delay is not associated with MSY2. Suppression subtractive hybridization will be used to define the mRNAs that are not bound by MSY2. These experiments will address an important post-transcriptional regulatory mechanism essential for both male and female gamete development.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD044449-03
Application #
6896223
Study Section
Reproductive Biology Study Section (REB)
Program Officer
Taymans, Susan
Project Start
2003-07-01
Project End
2008-04-30
Budget Start
2005-05-01
Budget End
2006-04-30
Support Year
3
Fiscal Year
2005
Total Cost
$320,963
Indirect Cost
Name
University of Pennsylvania
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Xu, Mingang; Medvedev, Sergey; Yang, Juxiang et al. (2009) MIWI-independent small RNAs (MSY-RNAs) bind to the RNA-binding protein, MSY2, in male germ cells. Proc Natl Acad Sci U S A 106:12371-6
Medvedev, Sergey; Yang, Juxiang; Hecht, Norman B et al. (2008) CDC2A (CDK1)-mediated phosphorylation of MSY2 triggers maternal mRNA degradation during mouse oocyte maturation. Dev Biol 321:205-15
Xu, Mingang; Hecht, Norman B (2008) MSY2 and polypyrimidine tract binding protein 2 stabilize mRNAs in the mammalian testis. Int J Androl 31:457-61
Yang, Juxiang; Morales, Carlos R; Medvedev, Sergey et al. (2007) In the absence of the mouse DNA/RNA-binding protein MSY2, messenger RNA instability leads to spermatogenic arrest. Biol Reprod 76:48-54
Yang, Juxiang; Medvedev, Sergey; Reddi, P Prabhakara et al. (2005) The DNA/RNA-binding protein MSY2 marks specific transcripts for cytoplasmic storage in mouse male germ cells. Proc Natl Acad Sci U S A 102:1513-8
Yang, Juxiang; Medvedev, Sergey; Yu, Junying et al. (2005) Absence of the DNA-/RNA-binding protein MSY2 results in male and female infertility. Proc Natl Acad Sci U S A 102:5755-60
Yu, Junying; Deng, Manqi; Medvedev, Sergey et al. (2004) Transgenic RNAi-mediated reduction of MSY2 in mouse oocytes results in reduced fertility. Dev Biol 268:195-206