Nucleic Acids Research, 2008, Vol. 36, No. 22 7161 Figure 2. KSRP binds to Pgk2 mRNA in the testis and in vitro to the F1 region of the Pgk2 mRNA and to PTBP2. (A) Messenger RNA detection by RT–PCR of puriﬁed germ cell mRNAs immunoprecipitated from mouse testis extracts with anti-KSRP. (B) A diagram of the three subclones (F1, F2 and F3) of the Pgk2 30-UTR. (C) KSRP selectively binds to the F1 region to the 30-UTR of Pgk2 mRNA. Testis extracts (20 mg) were incubated with [32P]-labeled transcripts generated by in vitro transcription from subclones (F1, F2 and F3) of the 30-UTR of Pgk2 mRNA as previously described (9). RNA–protein complexes were resolved in 4% polyacrylamide gels in 0.5 TBE buﬀer. Anti-KSRP was added to indicated extracts before the addition of the radiolabeled RNAs. (D) Western blot of endogenous t-KSRP coimmunoprecipitated with r-PTBP2 from testis extracts. His-tagged recombinant PTBP2 (20 mg) was incubated with His-tag aﬃnity resins and testis extract (1 mg). Lane 1, plus RNase A; lane 2, no RNAse; lane 3, antiactin control. Following centrifugation, pellets were solubilized and the proteins resolved in 10% SDS–PAGE. KSRP was detected by western blotting. (E) Recombinant t-KSRP and recombinant PTBP2 interact. GST-tagged r-KSRP was incubated with r-PTBP2. Following the addition of glutathione agarose beads and centrifugation, the pellets were solubilized, electrophoresed in 10% SDS–PAGE and PTBP2 was detected by western blotting. Lane 1, GST; lane 2, t-KSRP tagged with GST. PTBP2 (Figure 3B). Both proteins bind the F1 region of the 30-UTR of Pgk2 mRNA, yielding complexes with dif- ferent mobilities (Figure 3B, lanes 2 and 3). A distinct complex of slower electrophoretic mobility is seen when the two proteins are added together with F1 RNA (Figure 3B, lane 4). Addition of anti-KSRP (polyclonal or monoclonal) or anti-PTBP2 dissociates the KSRP– PTBP2–RNA complex (Figure 3B, lanes 5 and 6), demon- strating the presence of t-KSRP and PTBP2 in the same F1 RNA–protein complex. The 52-kDa KSRP is predominantly in the cytoplasm in mouse testis In somatic cells, the 75-kDa KSRP is involved in pre- mRNA splicing in the nucleus and in an mRNA decay pathway in the cytoplasm (17–26). Northern blotting of total RNA from brain and testes of prepuberal and adult mice detects one mRNA of 4 kb, in agreement with pub- lished reports (18). KSRP mRNA levels are higher in testes from 17-day-old mice than in the testes of 22-day-old and adult mice, suggesting it is more abundant in early-stage meiotic germ cells (Figure 4A, compare lanes 2–4). Although we only detect one KSRP mRNA of 4 kb in both testis and brain (Figure 4A) and an mRNA of this size encodes the 75 kDa KSRP in somatic cells (18), western blotting reveals two distinct KSRP protein bands of 75 and 52 kDa (Figure 4B). Separation of the testis into nuclear and cytoplasmic fractions reveals that the 75-kDa KSRP is nuclear (Figure 4B, lanes 1–3) while the majority of t-KSRP is in the cytoplasm (Figure 4B, lanes 4–6). Consistent with KSRP RNA levels (Figure 4A), higher amounts of t-KSRP are seen in the testes of prepuberal mice than in adults (Figure 4B, compare lanes 1 and 4 to lanes 3 and 6). In several RNA aﬃnity chromatography experiments with cytoplasmic testis extracts, t-KSRP was the only KSRP detected (9). The predominance of t-KSRP in the cyto- plasm of the testis suggests this 52-kDa isoform of KSRP could function as an mRNA-destabilizing protein in the testis (Figure 4B). t-KSRP is enriched in early stages of meiotic germ cells in mouse testes Two diﬀerent approaches, western blotting of proteins from highly puriﬁed populations of germ cells and