Characterization of SERCA2b Ca 2+–Mg 2+ ATPase mRNA decay by nuclear proteins

Abstract

Gene expression is controlled at several levels including mRNA decay. Sarco/endoplasmic reticulum Ca 2+–Mg 2+-ATPase isoform 2b (SERCA2b) is central to Ca 2+ signalling and homeostasis in several tissues. SERCA2b mRNA decay involves interactions between cis-acting elements in its 3′-region and trans-acting nuclear protein factors. In the presence of the protein factors, the synthetic capped and polyadenylated RNA fragment 2b1 (3444–3753) decays faster than other SERCA2b 3′-region fragments. Here we determined the minimum cis-acting destabilizing element in the decay and its interactions with the nuclear protein factors. The in vitro decay required ATP hydrolysis and Mg 2+ but not Ca 2+. The decay was directional from 3′ to 5′, and involved a novel 35b GC rich domain designated 2b1–4 corresponding to 3521–3555. The decay of 2b1 RNA was decreased by (a) competition with 2b1–4, (b) mutation of 2b1 to delete 2b1–4, and (c) depleting the extracts of destabilizing trans-acting factors using immobilized 2b1–4. To determine the minimal destabilizing elements 2b1–4 was divided into 7b domains A–E. Deleting AB, BC, CD or DE inactivated the destabilizing cis-acting element but deleting A, B, C, D or E had no effect. In electrophoresis mobility shift assays the nuclear protein extracts retarded the mobility of labeled uncapped 2b1 RNA without a poly A + tail. A positive co-operativity in the interactions was shown in protein concentration dependence of the shift and in the competition of 2b1–4 in inhibiting the mobility of 2b1 RNA. Based on further experiments, the domain CDE (3535–3555) was sufficient to compete with 2b1 RNA for the protein binding. Consistent with this competition, excess CDE RNA retarded the in vitro decay of 2b1 RNA. Thus the RNA decay required ATP hydrolysis and Mg 2+ but not Ca 2+, the minimum binding domain was in the sequence 3535–3555, and the decay may involve a multimeric protein complex.