Poster abstracts

Poster number 46 submitted by Sarah Grabinski

Ptbp2 is essential for germ cell development in male mice and exhibits tissue-specific patterns of RNA binding

Sarah E. Grabinski (Center for RNA Molecular Biology, Case Western Reserve University), Leah L. McCord (Center for RNA Molecular Biology, Case Western Reserve University), Thomas J. Sweet (Center for RNA Molecular Biology, Case Western Reserve University), Donny D. Licatalosi (Center for RNA Molecular Biology, Case Western Reserve University)

Abstract:
Ptbp2 is the brain and testis expressed paralog of polypyrimidine tract binding protein PTB. We have previously shown that Ptbp2 is essential for postnatal survival in mice, and functions predominantly as a silencer of alternative exon splicing in the embryonic brain to repress the expression of developmentally regulated ‘adult-enriched’ exons⁽¹⁾. To determine if tissue-restricted RNA binding proteins have the same or tissue-specific functions in RNA regulation in different biologic contexts we investigated the role of Ptbp2 in mouse testis. We generated Ptbp2 HITS-CLIP libraries from whole testis and performed comparative analyses with HITS-CLIP data generated from embryonic brain. This analysis identified RNA targets in which Ptbp2 binding is identical in brain and testis, and RNA targets where Ptbp2 exhibits tissue-specific differences in RNA binding suggestive of testis-specific and splicing-independent roles for Ptbp2 in RNA regulation. Using mice bearing a conditional ‘floxed’ allele of Ptbp2 (generously provided by Qin Li and Doug Black, UCLA) and the germ cell-specific Stra8-iCre driver⁽²⁾, we have generated mice with Ptbp2-null germ cells. Loss of Ptbp2 results in spermatogenic arrest following the production of haploid spermatids. Strikingly, chains of haploid spermatids (normally held together by intercellular bridges) coalesce to form giant multinucleate cells in Ptbp2-null testis. These observations indicate that RNA regulatory functions performed by Ptbp2 in germ cells are essential for mammalian germ cell development. To explore the functions of Ptbp2 in specific stages of germ cell development, we have developed a new germ cell purification strategy using a dual fluorescence RFP/GFP transgene⁽³⁾ and flow cytometry to isolate wild type and Ptbp2-null germ cells in each of the major stages of spermatogenesis. This approach will allow us to investigate the roles of Ptbp2 as germ cells progress through different stages of germ cell development, thus providing valuable insights into how a single RNA binding protein regulates its RNA targets in different cell types in an in vivo model of mammalian cell development.

References:
1)Licatalosi et al, 2012 Genes Dev 26, 1626-1642.
2)Sadate-Ngatchou et al, 2008 Genesis 46 738-742
3)De Gasperi et al, 2008 Genesis 46 308-317.

Keywords: spermatogenesis, CLIP, splicing