Brain RNAs undergo polyadenylation in injected Xenopus oocytes. (A) 3′ UTRs derived from ABP (AMPA binding protein), RCM3 (calmodulin), and ELK2 (ether a-go-go like, a potassium channel) mRNAs were synthesized in the presence of ³²P-UTP and injected into oocytes, some of which were incubated with progesterone. Total RNA was extracted, some of which was treated with oligo(dT) and RNase H and then analyzed by denaturing PAGE. (B) 3′ UTRs derived from δCaMKII, NGT (neuronal glutamate transporter), IGFII (insulin like growth factor II), MBP (myelin basic protein), Gabt1 (GABA transporter), and GlnA (glutamine synthetase) were injected into oocytes and analyzed for polyadenylation as in Figure 1. (C) 3′ UTRs derived from αCaMKII, CKB (creatine kinase B), C-CAM4 (cell–cell adhesion molecule 4), P2X2 (receptor for ligated-gated channel), PDE1 (phosphodiesterase 1), and γ tubulin (γTub) were injected into oocytes an examined for polyadenylation as in Figure 1.

Regimen for identifying brain mRNAs that undergo activity-dependent polyadenylation. (A) Schematic of αCaMKII mRNA denoting U-rich CPEs and polyadenylation hexanucleotide. A mutant 3′ UTR contains nucleotide substitutions in the CPEs (top). A portion of wild-type (WT) αCaMKII 3′ UTR was synthesized in vitro and injected into oocytes, some of which were treated with progesterone. The RNA was extracted and applied to poly(U) agarose. The beads were washed at 50°C and the RNA eluted at 60°C; αCaMKII RNA was detected by RT-PCR (middle). Wild-type (WT) and mutant (MUT) αCaMKII 3′ UTRs were injected into oocytes from three frogs; the RNA was analyzed as above. (B) A rat brain cDNA library (oligo(dT)-primed, directionally cloned downstream of the SP6 promoter and polylinker) is spread onto 25 plates so that each contains ~1000 colonies. The colonies are collected, plasmid DNA is isolated, linearized with NotI, and transcribed. The poly(A) tails are removed by oligo(dT)/RNase H and the RNA is injected into Xenopus oocytes that are induced to mature with progesterone. RNA extracted from the oocytes is applied to poly(U) agarose; the beads are washed at 50°C and the RNA eluted at 60°C. The newly polyadenylated brain RNA from each plate is combined and used to make biotin-labeled cRNA for probing an Affymetrix neurobiology microarray (chip RN U34 containing ~1200 sequences). Positive sequences are examined for the presence of a CPE and AAUAAA and some are then synthesized individually in vitro and retested for polyadenylation in injected oocytes. Some sequences are tested for activity-dependent polyadenylation in cultured hippocampal neurons.

Activity-dependent polyadenylation in neurons. (A) Cultured hippocampal neurons were untreated or stimulated with glutamate, KCl, or kainate. RNA was extracted and examined for polyadenylation as in Figure 1. (B) Protein from untreated synaptoneurosomes or those stimulated with glutamate for 10 min were Western blotted and probed for Map2, ABP, and synaptophysin. The average values from three experiments, ±SEM, are shown; the differences between the Map2 and ABP protein levels ± glutamate were statistically significant (p ≤ 0.05, Student’s t-test).