Nucleic Acids Research, 2008, Vol. 36, No. 22 7165 Figure 7. t-KSRP destabilizes mRNAs transfected into HeLa cells. (A) HeLa cells were cotransfected with pEGFP-C2-UTR (containing the entire 30-UTR of Pgk2) and vectors expressing t-KSRP or an N-truncated (cytoplasmic form) of PTBP2. Following actinomycin D addition, cells were collected at intervals, total RNAs were puriﬁed and mRNA levels were analyzed by northern blotting. b-Actin mRNA was used as a loading control. (B) The F3 subclone of the 30-UTR of Pgk2 and b-actin mRNA were used as nonbinding RNA incubation and loading controls, respectively. The F3 RNA was used in place of the F1 RNA because it contained the same polyadenylation signal as the transcript from pEGFP-C2-UTR. Each assay was repeated at least three times with essentially identical results. cell–speciﬁc DNA/RNA-binding protein, MSY2, stabilize many germ cell mRNAs (7), while the PTBP2 selectively stabilizes mRNAs such as the PGK 2 mRNA in the testis (9). Because many male germ cell mRNAs are sequestered into residual bodies, which are subsequently phagocytized by Sertoli cells, major eﬀorts to deﬁne mechanisms that degrade male germ cell mRNAs have not been under- taken. In contrast, in somatic cells, mRNA degradation events often dependent on AU-rich sequence elements (AREs) and proteins such as KSRP that bind to them have been well characterized (19,20). AREs are common cis-acting elements in eukaryotic transcripts that promote rapid mRNA turnover by recruit- ing KSRP-containing protein complexes to mRNAs (19). In addition to its originally described function(s) as a spli- cing factor (17,18), KSRP facilitates the degradation of short-lived mRNAs encoding proteins such cytokines, growth factors and protooncogenes through interactions with AREs in their 30-UTRs (19–26). The tethering of KSRP to HIV-1 mRNA constructs elicits rapid mRNA decay and a dramatic viral reduction by recruiting mRNA decay/machinery factors to the bound KSRP (24). Recently, the chicken protein, ZBP2, a homologue of KSRP, has been demonstrated to bind to sequences that control the subcellular localization of actin mRNAs (30,31). Three classes of AREs have been identiﬁed (32) based on the presence of one or more of the AUUUA motifs in their 30-UTRs (class I), multiple overlapping motifs in their 30-UTRs (class II) or no obvious AUUUA motifs in their 30-UTRs (class III). Although the 75-kDa KSRP is often the predominant KSRP in somatic cells, in the testis the 52-kDa KSRP, t-KSRP, is the primary isoform of KSRP in the cytoplasm (Figure 4B). Based on the existence of only one size of KSRP mRNA in testis, we believe that t-KSRP is not derived from an independent KSRP mRNA, but is either proteolytically cleaved from the somatic KSRP similar to the apoptotic cleavage of KSRP in liver (33) or t-KSRP is translated from an internal initiation site (methionine 207) in the 4-kb KSRP mRNA. Edman protein sequencing of t-KSRP suggests an N-terminus block (data not shown). Sequence analysis of 17 peptides from t-KSRP obtained following RNA aﬃnity chromato- graphy indicates that although t-KSRP lacks part of the ﬁrst RNA-binding domain, the remaining three KH RNA-binding domains are intact. We ﬁnd that t-KSRP is functionally active in mRNA binding, consistent with in vitro and in vivo studies demonstrating that the third and fourth KH domains of KSRP are suﬃcient for promoting mRNA decay in vivo (18,19). The functions of multifunctional proteins such as KSRP are often deﬁned by their subcellular location(s) (20). Numerous splicing studies with KSRP have been sup- ported by its nuclear localization, and a recent immuno- ﬂuorescent study concluded KSRP is almost exclusively nuclear in HeLa and neuroblastoma cells (27). Although KSRP shows ﬂexibility in recognition of RNA targets (34), binding of KSRP to AREs in 30-UTRs of mRNAs is indicative of cytoplasmic roles for KSRP (19–26). To accomplish both splicing and mRNA decay in somatic cells, KSRP would need to shuttle between the nucleus and cytoplasm as has been reported for other RNA-bind- ing proteins such hnRNP K, Translin and Trax (35,36). The N-terminus of KSRP likely contributes to its intracel- lular movement. The Xenopus homologue of KSRP, which lacks a 58-amino-acid segment near the N-terminus, is present in both the nucleus and the cytoplasm during oogenesis (37), consistent with our observation that in mouse testis the truncated t-KSRP is primarily in the cytoplasm, while the full-length KSRP is solely nuclear