Du Ling, Richter Joel D
University of Massachusetts Medical School, 373 Plantation St., Worcester, MA 01605, USA.
RNA. 2005 Sep;11(9):1340-7. doi: 10.1261/rna.2870505. Epub 2005 Jul 25.
Activity-dependent changes in protein synthesis modify synaptic efficacy. One mechanism that regulates mRNA translation in the synapto-dendritic compartment is cytoplasmic polyadenylation, a process controlled by CPEB, the cytoplasmic polyadenylation element (CPE)-specific RNA binding protein. In neurons, very few mRNAs are known CPEB substrates, and none appear to be responsible for the effects on plasticity that are found in the CPEB knockout mouse. These results suggest that the translation of other mRNAs is regulated by CPEB. To identify them, we have developed a functional assay based on the polyadenylation of brain-derived mRNAs injected into Xenopus oocytes, a surrogate system that carries out this 3' end processing event in an efficient manner. The polyadenylated RNAs were isolated by binding to and thermal elution from poly(U) agarose and identified by microarray analysis. Selected sequences that were positive for polyadenylation were cloned and retested for polyadenylation by injection into oocytes. These sequences were then examined for activity-dependent polyadenylation in cultured hippocampal neurons. Finally, the levels of two proteins encoded by polyadenylated mRNAs were examined in glutamate-stimulated synaptoneurosomes. These studies show that many mRNAs undergo activity-dependent polyadenylation in neurons and that this process coincides with increased translation in the synapto-dendritic compartment.
依赖于活性的蛋白质合成变化会改变突触效能。一种在突触树突区域调节mRNA翻译的机制是细胞质多聚腺苷酸化，这一过程由CPEB（细胞质多聚腺苷酸化元件（CPE）特异性RNA结合蛋白）控制。在神经元中，已知的CPEB底物mRNA很少，而且似乎没有一个能解释CPEB基因敲除小鼠中发现的对可塑性的影响。这些结果表明，其他mRNA的翻译受CPEB调控。为了识别它们，我们开发了一种基于注射到非洲爪蟾卵母细胞中的脑源性mRNA多聚腺苷酸化的功能检测方法，非洲爪蟾卵母细胞是一种能高效进行这种3'端加工事件的替代系统。通过与聚（U）琼脂糖结合并热洗脱来分离多聚腺苷酸化的RNA，并通过微阵列分析进行鉴定。对多聚腺苷酸化呈阳性的选定序列进行克隆，并通过注射到卵母细胞中重新检测其多聚腺苷酸化情况。然后在培养的海马神经元中检测这些序列的活性依赖性多聚腺苷酸化。最后，在谷氨酸刺激的突触神经小体中检测多聚腺苷酸化mRNA编码的两种蛋白质的水平。这些研究表明，许多mRNA在神经元中经历活性依赖性多聚腺苷酸化，并且这一过程与突触树突区域翻译增加相吻合。
