KSRP Modulation of GAP-43 mRNA Stability Restricts Axonal Outgrowth in Embryonic Hippocampal Neurons

The KH-type splicing regulatory protein (KSRP) promotes the decay of AU-rich element (ARE)-containing mRNAs. Although KSRP is expressed in the nervous system, very little is known about its role in neurons. In this study, we examined whether KSRP regulates the stability of the ARE-containing GAP-43 mRNA. We found that KSRP destabilizes this mRNA by binding to its ARE, a process that requires the presence of its fourth KH domain (KH4). Furthermore, KSRP competed with the stabilizing factor HuD for binding to these sequences. We also examined the functional consequences of KSRP overexpression and knockdown on the differentiation of primary hippocampal neurons in culture. Overexpression of full length KSRP or KSRP without its nuclear localization signal hindered axonal outgrowth in these cultures, while overexpression of a mutant protein without the KH4 domain that has less affinity for binding to GAP-43′s ARE had no effect. In contrast, depletion of KSRP led to a rise in GAP-43 mRNA levels and a dramatic increase in axonal length, both in KSRP shRNA transfected cells and neurons cultured from Ksrp+/− and Ksrp −/−embryos. Finally we found that overexpression of GAP-43 rescued the axonal outgrowth deficits seen with KSRP overexpression, but only when cells were transfected with GAP-43 constructs containing 3′ UTR sequences targeting the transport of this mRNA to axons. Together, our results suggest that KSRP is an important regulator of mRNA stability and axonal length that works in direct opposition to HuD to regulate the levels of GAP-43 and other ARE-containing neuronal mRNAs.

KH型剪接调控蛋白（KH-type splicing regulatory protein，KSRP）可促进含AU富集元件（AU-rich element，ARE）的mRNA的降解。尽管KSRP在神经系统中有所表达，但目前对其在神经元中的功能仍知之甚少。本研究探究了KSRP是否可调控含ARE的GAP-43 mRNA的稳定性。研究发现，KSRP可通过结合该mRNA的ARE使其不稳定，这一过程依赖其第四个KH结构域（KH4）的存在。此外，KSRP会与稳定因子HuD竞争结合上述序列。本研究还考察了KSRP过表达与敲低对体外培养原代海马神经元分化的功能影响。过表达全长KSRP或缺失核定位信号（nuclear localization signal）的KSRP，会抑制该培养体系中的轴突生长；而过表达缺失KH4结构域、对GAP-43的ARE结合亲和力降低的突变体蛋白，则无此效应。与之相反，敲低KSRP可使GAP-43 mRNA水平升高，且轴突长度显著增加，这一现象在转染KSRP短发夹RNA（shRNA）的细胞以及从Ksrp+/−和Ksrp−/−胚胎中分离培养的神经元中均存在。最后，研究发现过表达GAP-43可挽救KSRP过表达导致的轴突生长缺陷，但仅当细胞转染的GAP-43构建体含有可将该mRNA转运至轴突的3'非翻译区（3' UTR）序列时才有效。综上，本研究结果表明，KSRP是调控mRNA稳定性与轴突长度的重要因子，其通过与HuD直接拮抗，调控GAP-43及其他含ARE的神经元mRNA的水平。