7162 Nucleic Acids Research, 2008, Vol. 36, No. 22 Figure 3. KSRP and PTBP2 are present in the same complexes with the F1 region of the 30-UTR of Pgk2 mRNA. RNA gel shifts were performed with testis extracts as in (Figure 2B). (A) Lane 1, transcript F1 without testis extract; lane 2, transcript F1 with extract; lane 3, transcript F1 with extract and anti-PTBP2; lane 4, transcript F1 with extract, anti-PTBP2 and anti-KSRP. The position of one complex shifted by each antibody is shown by an arrow. (B) Recombinant t-KSRP and recombinant PTBP2 (1 mg) interact and form complexes with F1 RNA containing both proteins. Lane 1, transcript F1 without testis extract; lane 2, transcript F1 t-KSRP; lane 3, transcript F1 with PTBP2; lane 4, transcript F1 with PTBP2 and KSRP; lane 5, transcript F1 with PTBP2, t-KSRP and anti-KSRP (Abnova, Taiwan); lane 6, transcript F1 with PTBP2, t-KSRP and anti-PTBP2 (Abnova). immunohistochemistry, were used to establish the cellular and subcellular locations of KSRP in mouse testes. In western blots of germ cell extracts, t-KSRP was predomi- nant in early meiotic stages (preleptotene, leptotene/zygo- tene and pachytene spermatocytes) and was not detectable in round spermatids or condensing spermatids/residual bodies, suggesting it functions during meiosis, but not postmeiotically (Figure 4C). In contrast, the nuclear 75-kDa KSRP and the Pgk2 mRNA-stabilizing protein, PTBP2, increase during meiosis and are abundant in spermatids. To better determine the extent of expression of t-KSRP, western blots were prepared with extracts from several somatic tissues. The expression of t-KSRP appears limited because t-KSRP was detected in testis extracts (Figure 4D, lane 1), but not in brain, liver, kidney or lung extracts (Figure 4D, lanes 2–5). Using aﬃnity-puriﬁed antibodies that detect both KSRP isoforms in testes from adult mice by immunohis- tochemistry, KSRP is primarily present in the nuclei and cytoplasm of early and midstages of meiotic germ cells in stage VIII tubules, consistent with the northern and wes- tern blotting data (Figures 4 and 5, left side). In contrast, PTBP2 is more abundant in the nuclei and cytoplasm of later stage meiotic and postmeiotic germ cells in stage XI tubules (Figure 5, right side) (9). Based on the nuclear localization of KSRP and the predominance of t-KSRP in the cytoplasm (Figure 4B), we conclude that t-KSRP and not full-length KSRP is the primary (sole?) KSRP in the cytoplasm of early meiotic stage germ cells (Figure 5). KSRP destabilizes Pgk2 mRNA in extracts or cells in the presence of PTBP2 Since KSRP serves to destabilize mRNAs in diverse somatic cell types (19–26), we investigated whether t-KSRP could destabilize one of its target mRNAs, the Pgk2 mRNA, using three complementary approaches— in vitro decay assays, transient transfections and protein depletion assays. All conﬁrm a role for t-KSRP in male germ cell mRNA decay. To assess the role of t-KSRP in in vitro decay, 50-capped [32P]-labeled F1 or F2 transcript was incubated with testis extracts and increasing amounts of recombinant t-KSRP (Figure 6A). Addition of 50 nM of t-KSRP leads to com- plete F1 RNA degradation within 20 min of incubation (Figure 6A, lane 2), although only a modest RNA reduc- tion was seen in control testis extracts with up to 50 min of