Figure 4. Two isoforms of KSRP, KSRP and t-KSRP, are expressed from one mRNA in the testis, and t-KSRP is enriched in the cytoplasm of early-stage meiotic cells. (A) Northern blot of KSRP mRNA from brain and prepuberal and adult testes. Total RNAs (15 mg) were elec- trophoresed in 1% agarose–formaldehyde gels, transferred to Hybond- N + membranes and hybridized with a KSRP 30-UTR probe. Rehybridization of the blot with a coding region probe for actin served as a loading control. Lane 1, adult brain; lane 2, 17-day-old testis; lane 3, 22-day-old testes; lane 4, adult testes. (B) Western blot of KSRP in nuclear and cytoplasmic fractions of prepuberal and adult testes. Proteins (20 mg) extracted from puriﬁed nuclei and cytoplasm were separated by 10% SDS–PAGE and blotted onto polyvinylidene diﬂuoride membranes. Anti-KSRP was used at a dilution of 1:3000. Antiactin served as a loading control. Lanes 1–3 and 4–6 were extracts from nuclei and cytoplasm, respectively. Lanes 1 and 4, 17-day-old testis; lanes 2 and 5, 22-day-old testes; lanes 3 and 6, adult testes. (C) Western blot of KSRP, PTBP2 and actin in extracts of isolated populations of germ cells. Lane 1, preleptotene spermatocytes; lane 2, leptotene/zygotene spermatocytes; lane 3, early pachytene spermato- cytes; lane 4, pachytene spermatocytes; lane 5, round spermatids; lane 6, condensing spermatids/residual bodies. The preleptotene sper- matocytes, leptotene/zygotene spermatocytes and early pachytene sper- matocytes were isolated from the testes of 17-day-old mice, whereas the pachytene spermatocytes, round spermatids and condensing sperma- tids/residual bodies were isolated from the testes of adult mice. (D) Western blot of t-KSRP in tissue extracts. Lane 1, testis; lane 2, brain; lane 3, liver; lane 4, kidney; lane 5, lung protein extracts (30 mg) analyzed as in (B) with actin serving as a loading control. incubation (Figure 6A, lane 1). F1 RNA degradation increased when recombinant t-KSRP was increased to 100 nM (Figure 6A, lane 3). In contrast, no increased RNA degradation was seen when 100 nM t-KSRP was added to testis extracts containing the nonbinding F2 RNA (Figure 6A, compare lanes 6 and 7). As previously reported (9), addition of r-PTBP2 completely stabilized F1 RNA in incubations up to 50 min (Figure 6A, lane 5), but addition of up to 200 nM PTBP2 failed to prevent F1 Nucleic Acids Research, 2008, Vol. 36, No. 22 7163 Figure 5. Anti-KSRP selectively stains early-stage meiotic germ cells of the adult mouse testis. Immunostaining of testis sections from sexually mature CD-1 mice with (A) aﬃnity-puriﬁed anti-KSRP and (B) aﬃnity- puriﬁed anti-PTBP2. Anti-KSRP (Abnova, Taiwan) and anti-PTBP2 (from R. Darnell) were used at dilutions of 1:200. Magniﬁcation: 200, 400. Both antibodies only recognized their respective proteins on western blots. RNA destabilization when exogenous t-KSRP was added (Figure 6A, lane 4). These in vitro decay assays indicate that t-KSRP promotes RNA degradation of a target mRNA and suggest t-KSRP exerts a dominant eﬀect over PTBP2 in testis extracts under the assay con- ditions tested. The increased RNA degradation seen with increasing amounts of recombinant t-KSRP was also seen with endo- genous t-KSRP (Figure 6B). Radiolabeled F1 RNA was degraded more rapidly in testis extracts from prepuberal (17-day-old-mice) mice (which contain higher amounts of t-KSRP, Figure 4B) than in testis extracts from adult mice (8–10 weeks, Figure 6B, compare lanes 1 and 2), sug- gesting the more abundant t-KSRP in 17-day-old mice is consistent with the more rapid RNA degradation. The RNA degradation requires t-KSRP binding to RNA because no degradation was seen when the control nonbinding F2 RNA probe was incubated with pre- puberal or adult extracts (Figure 6B, compare lanes 3 and 4). In vitro decay assays with extracts depleted of KSRP by immunoprecipitation provide additional evi- dence for the destabilizing role for endogenous t-KSRP (Figure 6C). The stability of radiolabeled F1 RNAs in these assays was increased in adult testis extracts that had been depleted of KSRP (Figure 6C, compare lanes 1 and 2). Again this required RNA binding as no diﬀerences were seen with the F2 RNA (Figure 6C, compare lanes 3 and 4). To determine whether t-KSRP can also destabilize mRNAs and be dominant to PTBP2 in living cells, an EGFP construct containing the 30-UTR of Pgk2 mRNA was cotransfected into HeLa cells with t-KSRP and with or without PTBP2 (Figure 7). For PTBP2 expression, an N-truncated form of PTBP2 was used because this mod- iﬁcation targets PTBP2 to the cytoplasm, whereas full- length PTBP2 contains a functional nuclear localization