7158 Nucleic Acids Research, 2008, Vol. 36, No. 22 of proteins that function as nuclear splicing factors, direct Internal Ribosome Entry Site (IRES)-directed translation initiation and stabilize mRNAs in the cytoplasm (11–17). In vitro assays reveal that PTBP2 increases the stability of Pgk2 mRNA in both testis extracts and in transfected HeLa cells, suggesting that PTBP2 helps maintain the in vivo stability of the Pgk2 mRNA by binding to a reg- ulatory element (9). To begin to identify proteins that destabilize germ cell mRNAs, RNA aﬃnity chromatography was used to screen testis cytoplasmic extracts with the 30-untranslated region (UTR) of Pgk2 mRNA. Among several RNA- binding proteins, a novel 52-kDa isoform of the multi- functional KH-type splicing regulatory protein (t-KSRP) was identiﬁed (9). An 75-kDa KSRP that is ubiquitously expressed has been implicated in transcriptional regula- tion, splicing and mRNA decay in somatic tissues and cultured cells (17–26). Here we demonstrate that t-KSRP binds to an AU-rich sequence in the 30-UTR of Pgk2 mRNA and destabilizes the Pgk2 mRNA. Moreover, the destabilization of Pgk2 mRNA occurs when t-KSRP and PTBP2 are present together in complexes bound to the 30-UTR of Pgk2 mRNA. MATERIALS AND METHODS Plasmid construction and expression of the 52-kDa KSRP Mass spectrometry sequence analysis of a 52-kDa pro- tein puriﬁed from an adult testis cytoplasmic extract by RNA aﬃnity chromatography identiﬁed 17 peptides mapping to KSRP. The peptides ranged from amino acid 218–684 (9, Table 1). In western blots, an antibody against the C-terminus of the 75-kDa KSRP (DB-KS, Figure 4B) recognized both the testicular KSRPs, suggest- ing that the 52-kDa KSRP isoform (t-KSRP) is an N-terminally truncated form of KSRP. Based on the MS-TOF sequences, the western blotting (see below), and the 52-kDa electrophoretic mobility of t-KSRP, a recombinant t-KSRP was synthesized starting at methio- nine 207 and terminating at amino acid 748 (the last amino acid of the 75-kDa KSRP). This recombinant N-truncated KSRP was synthesized because it is not known if the smaller KSRP isoform is translated from an internal methionine (at amino acid 207) or is a cleavage product of the 75-kDa KSRP. Based on mass spectro- metry and estimated protein size, the recombinant protein synthesized is very close in size to a protein containing a peptide starting at amino acid 218 (Table 1). The 52-kDa protein, t-KSRP207-748 (NM_010613), was ampliﬁed by reverse transcription–polymerase chain reaction (RT–PCR) from adult mouse testis total RNA and subcloned into pET-42a (Novagen, San Diego, CA). Recombinant t-KSRP was expressed in BL21-CodonPlus (DE3)-RP cells (Stratagene, La Jolla, CA), puriﬁed with a Glutathione S-transferase (GST) puriﬁcation kit and, when necessary, digested with thrombin to remove the GST tag. For the transfection studies, the PCR product of t-KSRP (207–748 of full-length KSRP) was digested with HindIII and SalI and cloned into the HindIII/SalI Table 1. MALDI-TOF analysis for 52 kDa KSRP Peptide sequence Location in 75 kDa KSRP (amino acid number) GGPPGQFHDNANGGQNGTV QEIMIPAGK AGLVIGKGGETIK GGETIKQLQER MILIQDGSQNTNVDKPLR ERDQGGFGDR DQGGFGDR VGGGIDVPVPR HSVGVVIGR IQNDAGVR IAHIMGPPDR IINDLLQSLR GRGQGNWGPPGGEMTFSIPTHK AINQQTGAFVEISR GSPQQIDHAK AWEEYYK IGQQPQQPGAPPQQDYTK QAQVATGGGPGAPPGSQPDYSA AWAEYYR 218–245 246–258 253–263 268–285 306–315 308–315 322–332 333–341 349-356 370–379 386–395 415–436 450–463 480–489 622–628; 648–654 630–647 656–684 site of pEGFP-C2 (Clontech, Palo Alto, CA) to generate the expression plasmid, pEGFP-KSRP. Semiquantitative RT–PCR Total RNA was extracted from the testes of 17-day-old, adult, wild-type mice with Trizol (Invitrogen, Carlsbad, CA), residual genomic DNA was removed with the TurboDNA free kit (Ambion, Austin, TX) and the amount of Pgk2 mRNA was quantitated using qualitative RT–PCR. First-strand complementary DNA was synthe- sized with random hexamer primers and Superscript III reverse transcriptase following the manufacturer’s instruc- tions (Invitrogen). Qualitative PCR reactions were per- formed with SYBRgreen PCR Master reagents and the 7900HT sequence detector (PE Applied Biosystems, Foster City, CA). The expression level of Pgk2 mRNA was normalized to actin mRNA. Nuclear run-on assays Nuclei were isolated, and nuclear run-on reactions were performed as previously described (7). Brieﬂy, a nuclear suspension (100 ml) was mixed with 100 ml of 2 reaction buﬀer (10 mM Tris–HCl, pH 8.0, 5 mM MgCl2, 0.3 M KCl, 5 mM Dithiothreitol (DTT), 1 mM Adenosine triphosphate (ATP), 1 mM Cytidine triphosphate (CTP) and 1 mM Guanosine triphosphate (GTP) and 10 ml of [P32]-UTP and incubated at 308C for 30 min. The reaction was terminated by the addition of 6 ml of 250 mM CaCl2 and 6 ml of RNase-free DNase I (25 mg/ml). After incu- bation for 10 min at 298C, the samples were incubated with proteinase K (10 mg/ml) at 428C for 30 min, and RNA was puriﬁed with Trizol (Invitrogen) before hybrid- ization to DNAs bound to nitrocellulose membranes. Hybridizations were carried out for 24 h at 658C in Quik hybridization solution (Stratagene), and hybridization signals were detected by PhosphorImager (Amersham Biosciences, Piscataway, NJ).