Gld2-catalyzed 3' monoadenylation of miRNAs in the hippocampus has no detectable effect on their stability or on animal behavior.. Gld2-catalyzed 3' monoadenylation of miRNAs in the hippocampus has no detectable effect on their stability or on animal behavior.

Gld2, a noncanonical cytoplasmic poly(A) polymerase, interacts with the RNA binding protein CPEB1 to mediate polyadenylation-induced translation in dendrites of cultured hippocampal neurons. Depletion of Gld2 from the hippocampus leads to a deficit in long-term potentiation evoked by theta burst stimulation. At least in mouse liver and human primary fibroblasts, Gld2 also 3' monoadenylates and thereby stabilizes specific miRNAs, which enhance mRNA translational silencing and eventual destruction. These results suggest that Gld2 would be likely to monoadenylate and stabilize miRNAs in the hippocampus, which would produce measurable changes in animal behavior. We now report that using Gld2 knockout mice, there are detectable alterations in specific miRNA monoadenylation in the hippocampus when compared to wild type, but that these modifications produce no detectable effect on miRNA stability. Moreover, we surprisingly find no overt change in animal behavior when comparing Gld2 knockout to wild-type mice. These data indicate that miRNA monoadenylation-mediated stability is cell type-specific and that monoadenylation has no measurable effect on higher cognitive function. Overall design: To assess the involvement of Gld2 in nontemplated nucleotide addition to miRNAs, hippocampi from six WT and six Gld2 KO male mice were isolated and cDNA libraries were constructed from gel-isolated RNAs between 18 and 32 bases in length, which primarily include miRNAs.

Gld2作为一种非经典胞质聚腺苷酸聚合酶（noncanonical cytoplasmic poly(A) polymerase），可与RNA结合蛋白CPEB1（胞质多聚腺苷酸化元件结合蛋白1，Cytoplasmic Polyadenylation Element Binding Protein 1）相互作用，介导培养海马神经元树突内由多聚腺苷酸化诱导的翻译过程。从海马体中耗竭Gld2会导致θ爆发刺激（theta burst stimulation）诱导的长时程增强（long-term potentiation, LTP）出现功能缺陷。至少在小鼠肝脏与人类原代成纤维细胞（human primary fibroblasts）中，Gld2还可对微小RNA（microRNA, miRNA）进行3'端单腺苷酸化修饰，借此稳定特定miRNAs，进而增强mRNA的翻译沉默及最终降解过程。上述结果提示，Gld2或可在海马体中对miRNAs进行单腺苷酸化修饰并维持其稳定性，从而对动物行为产生可检测到的改变。本研究现报道，与野生型（wild type, WT）小鼠相比，Gld2基因敲除（Gld2 knockout, KO）小鼠的海马体中，特定miRNAs的单腺苷酸化修饰存在可检测到的变化，但此类修饰并未对miRNAs的稳定性产生可检测到的影响。此外，本研究意外发现，与野生型小鼠相比，Gld2基因敲除小鼠并未出现明显的动物行为改变。上述数据表明，miRNA单腺苷酸化介导的稳定性调控具有细胞类型特异性，且单腺苷酸化修饰对高等认知功能无可检测到的影响。实验整体设计：为评估Gld2在miRNAs的非模板核苷酸添加过程中的作用，本研究分离了6只WT雄性小鼠与6只Gld2 KO雄性小鼠的海马体，并从长度介于18至32个碱基的凝胶分离RNA中构建cDNA文库（complementary DNA library），此类RNA主要包含miRNAs。