7160 Nucleic Acids Research, 2008, Vol. 36, No. 22 Figure 1. (A) Quantitation of Pgk2 mRNA levels in prepuberal and adult testes. RT–PCR was used to measure Pgk2 mRNA levels in total RNA prepared from prepuberal (17 days) and adult testes. (B) Nuclear run-on assays were used to determine the transcriptional rate for Pgk2, Tnp2 and Gapdh mRNAs. At the completion of the nuclear run reactions, RNA was puriﬁed and hybridized to DNA probes ﬁxed to membranes. Lane 1, nuclei from 17-day-old mice; lane 2, nuclei from adult mice (6- to 8-weeks old). (Figure 1B). As reported for run-on transcription assays with nuclei from pachytene spermatocytes and round sper- matids (29), similar levels of Pgk2 mRNA transcription were detected in nuclei isolated from the testes of prepub- eral (17 day old) and adult mice (Figure 1B). Given that the proportion of spermatocytes in prepuberal testes from 17-day-old mice is similar to that of spermatocytes plus round spermatids in adult testes, these results suggest that the increase in Pgk2 mRNA levels in the adult testis reﬂects an mRNA buildup resulting from stabilization of Pgk2 mRNA in late-stage meiotic and postmeiotic germ cells (9) rather than an increase in transcription of the Pgk2 gene. KSRP is present in a Pgk2 mRNA–protein complex in testis extracts RNA aﬃnity chromatography with a biotin-labeled RNA representing the 30-UTR of Pgk2 and adult testis cytoplasmic extracts detected several proteins including an 52-kDa isoform of the RNA-binding protein KSRP (9). In somatic cells, a ubiquitously expressed 75-kDa KSRP has been implicated in transcriptional regulation, splicing and mRNA decay (17–26). Although the testis contains both KSRP isoforms, the 75-kDa KSRP was not detected in repeated chromatography experiments with cytoplasmic extracts consistent with the 52-kDa variant being the sole isoform of KSRP in the cytoplasm (see below). To determine whether KSRP is bound to Pgk2 mRNA in the testis, RNA–protein complexes were immunopreci- pitated with anti-KSRP from adult testes extracts. Following puriﬁcation, RT–PCR was used to assay the precipitated RNAs for Pgk2 mRNA and two control highly abundant germ cell mRNAs that lack known KSRP binding sites. Pgk2 mRNA was immunoprecipi- tated with anti-KSRP (Figure 2A), while protamine 2 and transition protein 2 mRNAs were not, demonstrating the selective immunoprecipitation of a KSRP–Pgk2 mRNA complex. None of the three mRNAs was precipi- tated with preimmune serum. KSRP binds to the F1 region of the 3’-UTR of Pgk2 mRNA To determine where KSRP binds in the 30-UTR of the Pgk2 mRNA, gel shifts were performed with adult testis cytoplasmic extracts and three [P32]-labeled RNA sub- cloned probes of the entire 30-UTR [F1 (93 nt), F2 (80 nt) and F3 (86 nt) (Figure 2B) (9)]. An RNA–protein complex was detected with F1 RNA (Figure 2C, lane 2) but not with the F2 or F3 RNA. In gel shifts with sub- clones of the F1-binding element, KSRP binds to a 34-nt region adjacent to the coding region (data not shown). Moreover, this binding to a speciﬁc region of the Pgk2 mRNA was speciﬁc because no binding was detected with the F1 subclone or any other part of the 30-UTR of Pgk1 mRNA (data not shown). PTBP2 also binds directly to the F1 region of the 30-UTR of the Pgk2 mRNA (9). t-KSRP and PTBP2 interact In somatic cells, PTBP and the 75-kDa KSRP have been demonstrated to interact as components of nuclear spli- cing complexes (11,17) and to bind together to the 30-UTRs of mRNAs they regulate in the cytoplasm (15). To assess whether the 52-kDa KSRP interacts with PTBP2 in male germ cells, two approaches were taken. First, His-tagged recombinant PTBP2 was added to and precipitated from testis extracts that had been pretreated with RNase A. Western blotting of the proteins that were precipitated with anti-KSRP detected the 52-kDa KSRP (Figure 2D), suggesting a protein–protein interaction between t-KSRP and PTBP2. To conﬁrm a direct interaction between PTBP2 and t-KSRP (see the Materials and Methods section for the t-KSRP cloning and expression criteria and methodolo- gies), puriﬁed recombinant PTBP2 was incubated with puriﬁed recombinant GST-tagged t-KSRP and anti- GST. PTBP2 was precipitated in the presence of GST- tagged t-KSRP, but not when GST was substituted for GST-tagged t-KSRP, establishing a direct protein–protein interaction between PTBP2 and t-KSRP (Figure 2E). Thus, t-KSRP can bind both to the F1 region of the 30-UTR of Pgk2 mRNA and to PTBP2. A complex of PTBP2 and t-KSRP binds to F1 RNA Both t-KSRP and PTBP2 selectively bind to a 93-nt region (F1) of the 30-UTR of Pgk2 mRNA (9, Figure 2C). To determine whether t-KSRP and PTBP2 are bound in the same RNA–protein complex, supershift assays using anti- PTBP2 or anti-KSRP were performed with radiolabeled F1 RNA and testis extracts (Figure 3A) or with recombi- nant t-KSRP and PTBP2 (Figure 3B). The addition of anti-KSRP to testicular extracts supershifts several F1 RNA–protein complexes from testicular extracts, indicat- ing t-KSRP is a component of multiple complexes with diﬀerent electrophoretic mobilities (Figure 3A, lane 4). Anti-PTBP2 supershifts multiple complexes, indicating its presence in RNA–protein complexes containing t-KSRP (Figure 3A, lane 3). The presence of both t-KSRP and PTBP2 in the same RNA–protein complex(es) is also seen in gel shifts per- formed with recombinant t-KSRP and recombinant