Figure 6.

t-KSRP destabilizes RNAs containing the 3′-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 30°C 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 25°C. (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.