Published online 10 November 2008 Nucleic Acids Research, 2008, Vol. 36, No. 22 7157–7167 doi:10.1093/nar/gkn800 A cytoplasmic variant of the KH-type splicing regulatory protein serves as a decay-promoting factor for phosphoglycerate kinase 2 mRNA in murine male germ cells Mingang Xu 1 , John R. McCarrey2 and Norman B. Hecht1,* 1Center for Research on Reproduction and Women’s Health, University of Pennsylvania School of Medicine, 1310 Biomedical Research Building, 421 Curie Boulevard, Philadelphia, PA 6080 and 2Department of Biology, University of Texas at San Antonio, San Antonio, TX 78249, USA Received August 25, 2008; Revised October 8, 2008; Accepted October 12, 2008 ABSTRACT Phosphoglycerate kinase 2 (PGK2) is a germ cell- specific protein whose mRNA is translationally regulated in the mammalian testis. Using RNA affi- nity chromatography with the 3’-untranslated region (UTR) of Pgk2 mRNA and adult testis extracts, sev- eral associated proteins including a novel isoform of the AU-rich element RNA-binding protein and KH- type splicing regulatory protein (KSRP) were identi- fied. KSRP, a protein of 75 kDa, is widely expressed in somatic and germ cells where it is pri- marily nuclear. In addition to the 75-kDa KSRP, a 52-kD KSRP, t-KSRP, is present in the cytoplasm of a subpopulation of germ cells. t-KSRP binds directly to a 93-nt sequence (designated the F1 region) of the 3’-UTR of the Pgk2 mRNA and desta- bilizes Pgk2 mRNA constructs in testis extracts and in transfected cells. We conclude that this testicular variant of the multifunctional nucleic acid–binding protein, KSRP, serves as a decay-promoting factor for Pgk2 mRNA in male germ cells. INTRODUCTION Phosphoglycerate kinase (PGK) is a highly conserved and widely expressed glycolytic enzyme. Mammals express two functional PGKs, PGK1, an X-linked ubiquitous protein expressed in somatic cells, oogenic cells and premeiotic and meiotic male germ cells, and a testis-speciﬁc isoform, PGK2, expressed in postmeiotic male germ cells and sper- matozoa. The latter is believed to have evolved by retro- position to compensate for the loss of PGK1 expression following X-chromosome inactivation during spermato- genesis (1). In mice, Pgk2 mRNA is ﬁrst detected in early stages of meiotic spermatocytes and dramatically increases in postmeiotic spermatids, while the PGK2 pro- tein is not detected until many days later in late-stage spermatids (2–4). Microarray studies have conﬁrmed the posttranscriptional regulation of Pgk2, demonstrating that Pgk2 mRNAs are initially detected as ribonucleopro- teins in meiotic spermatocytes and then move onto poly- somes in later stage haploid germ cells (5). This temporal separation of Pgk2 transcription and translation for up to 2 weeks requires both long-term Pgk2 mRNA stabilization and translational activation/degradation, suggesting that trans-acting factors are important in controlling Pgk2 expression. In the mammalian testis, posttranscriptional regulation of mRNA is essential for the sequential expression of pro- teins, especially in haploid germ cells because of the termi- nation of transcription as spermatids diﬀerentiate (6). The testis contains many RNA-binding proteins, which regu- late posttranscriptional events including mRNA proces- sing, transport, localization, stability and translation. The germ cell–speciﬁc DNA/RNA-binding Y-box protein, MSY2, and the polypyrimidine tract binding protein 2 (PTBP2) are two proteins that play important roles in germ cell mRNA stabilization (7–9). MSY2 is one of the most abundant RNA-binding proteins in male germ cells, constituting 0.7% of total soluble protein. It functions as a global stabilizer/translational suppressor of many germ cell mRNAs. MSY2 speciﬁcally recognizes a popu- lation of germ cell mRNAs by binding in the nucleus to a consensus promoter sequence present in their genes thereby linking the nuclear events of transcription with the cytoplasmic processes of mRNA storage and stabiliza- tion (10). Deletion of MSY2 by gene targeting leads to precocious translation and destabilization/loss of many mRNAs (7,8). PTBP2, a second protein that stabilizes testicular mRNAs, belongs to a multifunctional family *To whom correspondence should be addressed. Tel: +1 215 898 0144; Fax: +1 215 573 7627; Email: nhecht@mail.med.upenn.edu 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.