7166 Nucleic Acids Research, 2008, Vol. 36, No. 22 (Figure 4). The abundance and cytoplasmic location of t-KSRP and the nuclear location of the 75-kDa KSRP support a posttranscriptional mRNA regulatory role for t-KSRP in the mammalian testis. We have identiﬁed a testicular variant of the KSRP, t-KSRP, a protein that we believe facilitates the decay of mRNAs such as Pgk2 mRNA in meiotic germ cells. In contrast to many of the rapidly degraded mRNAs recognized by KSRP in somatic cells, t-KSRP binds to a 93-nt AU-rich class III region in the 30-UTR of the Pgk2 mRNA, a germ cell mRNA that may be short- lived mRNA during meiosis, but becomes a long-lived stable mRNA in postmeiotic germ cells. This transition appears to be facilitated by another RNA-binding protein in the testis, PTBP2, which functions to maintain Pgk2 mRNA levels (9). Thus, upon reduction of levels of t-KSRP during the transition from spermatocytes to sper- matids, the dominant Pgk2 mRNA decay-promoting activity is lost and the transcript-stabilizing activity of PTBP2 prevails, leading to a dramatic increase in the level of Pgk2 transcript in round spermatids in the absence of any increase in transcription rate of the Pgk2 gene. In vitro assays and transfection studies indicate the PTBP2 stabilizes the Pgk2 mRNA by direct binding to the F1 region of the 30-UTR of the Pgk2 mRNA, the same part of the 30-UTR recognized by t-KSRP. Since t-KSRP and PTBP2 interact and both recognize the same RNA-binding region of the Pgk2 mRNA, this raises the question how t-KSRP destabilizes mRNAs in the presence of PTBP2. Although the mechanism resulting in the appar- ent dominance of the destabilizing eﬀect of t-KSRP over the stabilizing eﬀect of PTBP2 is not known, we do know that the two proteins interact and form F1 RNA–protein complexes that contain both proteins. In the nuclei of WERI-1 retinoblastoma cells, the binding of full-length KSRP to RNA requires the adjacent binding of PTBP2, and the interaction of KSRP, PTBP2 and other factors facilitates the cooperative assembly of an hnRNP complex for splicing (11,17). In the cytoplasm, less is known about the functional interactions between the KSRP and PTBP proteins. As we propose for t-KSRP and PTBP2 in the testis, binding of KSRP and PTBP1 is believed to control the decay and stability of human inducible nitric oxide synthase mRNA, but the mechanism of their molecular interactions is not known (15,21). The apparent dominance of t-KSRP over PTBP2 may reﬂect in vivo RNA binding diﬀerences among t-KSRP, PTBP2 and accessory proteins/factors. Alternatively, pro- tein availability may be important because during early meiosis when Pgk2 mRNA levels are lowest, t-KSRP is most abundant and PTBP2 levels are low. t-KSRP levels decrease in later meiotic germ cells and round spermatids and is not detectable in condensing spermatids (Figure 4C), consistent with the 20-fold increase of Pgk2 mRNA in the testes of adult mice, resulting at least in part from the absence of t-KSRP in later stage postmeiotic germ cells (Figure 1). Protein variants (isoproteins) are especially common in male germ cells. The testis expresses isoforms of many proteins including lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, phosphoglycerate mutase, aldolase, pyruvate dehydrogen- ase E1 alpha, PGK and even KSRP (4). The isoprotein PGK2 is believed to have evolved to replace PGK1 because PGK1 becomes inactivated during meiotic sex- chromosome inactivation. Other isoproteins have been proposed to optimize metabolic activities unique to germ cells. The temporal synthesis patterns of many of these proteins require delayed mRNA utilization, suggesting that t-KSRP and PTBP2 are likely to regulate the cyto- plasmic steady-state levels of numerous germ cell mRNAs in addition to Pgk2 mRNA. Sequence analysis of mRNAs precipitated by anti-PTBP2 and anti-KSRP includes Tex27, Ddc8 and Spata6, genes whose mRNAs are trans- lationally regulated similar to Pgk2 mRNA (data not shown) (5). In summary, Pgk2 mRNA is ﬁrst detectable in early meiosis in spermatocytes and increases dramatically in spermatids (2–4, Figure 1). t-KSRP is abundant in early meiosis and markedly decreases, becoming undetectable in postmeiotic male germ cells (Figures 4 and 5). The decline of t-KSRP and increase of PTBP2 are concomitant with the increase in Pgk2 mRNA levels. We speculate that in the cytoplasm of early meiotic spermatocytes, t-KSRP promotes the decay of Pgk2 mRNA and as t-KSRP levels decrease, Pgk2 mRNA levels rise from continued transcription and stabilization by protein complexes con- taining PTBP2 in late-stage meiotic spermatocytes and postmeiotic spermatids. Many additional proteins are likely essential for these processes. In somatic tissues, KSRP stimulates mRNA decay by recruiting a decay-pro- moting exosomal complex to target mRNAs (19). In testis extracts, t-KSRP interacts with exosomal proteins such as EXOSC7 (Rrp42p) and Rrp40p (data not shown). Thus, a number of trans-acting factors will contribute to the syn- thesis/stabilization/destabilization of the Pgk2 mRNA during spermatogenesis. Recently, a number of additional germ cell mRNAs have been demonstrated to exhibit tem- poral RNP/polysomal regulation similar to Pgk2 mRNA (5). The possibility that they represent additional t-KSRP target mRNAs that undergo a coordinated regulation merits investigation. ACKNOWLEDGEMENTS We thank Dr R.B. Darnell (Rockefeller University, New York) for anti-PTBP2 and Dr D.L. Black (University of California, Los Angeles) for anti-KSRP (ab5 and DB-KS). FUNDING National Institutes of Health (grant HD 28832). Conﬂict of interest statement. None declared. REFERENCES 1. McCarrey,J.R. and Thomas,K. (1987) Human testis-speciﬁc PGK gene lacks introns and possesses characteristics of a processed gene. Nature, 326, 501–505. 2. Bluthmann,H., Cicurel,L., Kuntz,G.W., Haedenkamp,G. and Illmensee,K. (1982) Immunohistochemical localization of mouse