7164 Nucleic Acids Research, 2008, Vol. 36, No. 22 Figure 6. t-KSRP destabilizes RNAs containing the 30-UTR of Pgk2 mRNA. (A) t-KSRP destabilizes RNA constructs in testis extracts, and recombinant PTBP2 cannot prevent the destabilization. 5’-capped and 3’-polyadenylated radiolabeled F1 or F2 transcript was incubated at 308C with testis extracts with or without recombinant t-KSRP (100 or 200 nM) or PTBP2 (200 nM). RNA was puriﬁed and analyzed by gel electrophoresis followed by autoradiography. Gels were quantitated by phosphorimaging. The amount of RNA at the beginning of the reaction was set at 100%. All the decay assays were performed at least twice. (B) F1 RNAs are degraded more rapidly in extracts from 17-day-old mice than from adults. Decay assays were performed as in (A) with prepuberal (extracts from testes of 17-day-old mice) or adult extracts except that incubation temperature was set at 258C. (C) Depletion of KSRP from testis extracts leads to the stabilization of F1 RNA. Decay assays were performed as in (A) with cytoplasmic extracts from which KSRP was depleted. signal directing PTBP2 to nuclei (9). Previous studies have demonstrated that the full-length and truncated forms of PTBP2 are functionally indistinguishable (9). Consistent with the RNA incubations in testis extracts (Figure 6), the transfected RNA is less stable in the transfected cells expressing t-KSRP (Figure 7A, lane 2). Similar to the ﬁndings with testis extract incubations, the radiolabeled RNA is stabilized by the expression of PTBP2 (Figure 7A, lane 4), but cotransfection of PTBP2 and t-KSRP (the expression of both was conﬁrmed by western blotting, data not shown) leads to RNA destabilization (Figure 7A, lane 3), The RNA destabilization required RNA binding because there was no destabilization when a control nonbinding F3 RNA was transfected with t-KSRP (Figure 7B, compare lanes 1 and 2). Assays for b-actin mRNA established equal RNA loading per lane and speciﬁcity for the RNA destabilization of t-KSRP (see b-actin lanes in Figure 7A and B). We conclude that t-KSRP destabilizes RNAs such as the Pgk2 mRNA in cells and overrules the stabilizing ability of PTBP2 in transfected cells and in testis extracts (Figures 6 and 7). DISCUSSION Here we report the existence of a cytoplasmic isoform of KSRP (t-KSRP) that binds to a class III AU-rich sequence in the 30-UTR of Pgk2 mRNA and destabilizes the RNA in in vitro and cellular assays. t-KSRP interacts with and is a component of RNA–protein complexes con- taining PTBP2, another multifunctional RNA-binding protein that serves to stabilize RNAs in the cytoplasm of germ cells. When both PTBP2 and t-KSRP are present, t-KSRP appears to dominate and Pgk2 mRNA is destabilized. Protein synthesis is dependent on mRNAs whose steady-state levels are regulated by a combination of tran- scription, stabilization and decay. There is a growing awareness that posttranscriptional events play major reg- ulatory roles in mRNA metabolism and function in eukar- yotic cells. Although microarray studies provide great insight into the temporal and cellular transcription of the testicular transcriptome (5), mRNA stabilization is an essential regulatory mechanism in haploid diﬀerentiat- ing germ cells (6). Proteins such as the abundant germ