Dobson Tara, Minic Angela, Nielsen Kirsten, Amiott Elizabeth, Krushel Les
Department of Pharmacology, University of Colorado Health Sciences Center Denver, CO 80262, USA.
Nucleic Acids Res. 2005 May 20;33(9):2929-41. doi: 10.1093/nar/gki605. Print 2005.
Translational regulation of the dendritically localized mRNA encoding for the neurotrophin receptor TrkB has important ramifications for synaptic function. We examined whether the TrkB mRNA is translated through an internal initiation entry site (IRES). The human TrkB 5' leaders are derived from the use of alternative promoters and alternative splicing, but all 5' leaders share a common exon. Insertion of a full-length 5' leader, as well as the common exon into the intercistronic region of a dicistronic luciferase construct, yielded luciferase activity generated from the second cistron that was either equivalent or higher than that observed from the encephalomyocarditis virus IRES. Moreover, inhibiting cap-dependent translation ex vivo and in in vitro lysates had only a minimal effect on the translation of mRNA containing the TrkB 5' leader. Dissecting the 5' leader showed that the IRES is located in the exon common to all TrkB 5' leaders. Moreover, six regions ranging from 2 to 25 nt were identified that either promoted or inhibited IRES activity. Taken together, these results suggest that the 5' leader of the human TrkB mRNA contains multiple cis-elements that regulate internal initiation of translation and that this mechanism may contribute significantly to the translation of the TrkB mRNA in neuronal dendrites.
编码神经营养因子受体TrkB的树突定位mRNA的翻译调控对突触功能具有重要影响。我们研究了TrkB mRNA是否通过内部起始进入位点（IRES）进行翻译。人类TrkB的5'前导序列源自使用不同的启动子和可变剪接，但所有5'前导序列都共享一个共同的外显子。将全长5'前导序列以及共同外显子插入双顺反子荧光素酶构建体的顺反子间区域，产生了来自第二个顺反子的荧光素酶活性，该活性与从脑心肌炎病毒IRES观察到的活性相当或更高。此外，在体外和体外裂解物中抑制帽依赖性翻译对含有TrkB 5'前导序列的mRNA的翻译只有最小的影响。对5'前导序列进行剖析表明，IRES位于所有TrkB 5'前导序列共有的外显子中。此外，还鉴定出了六个长度从2到25个核苷酸的区域，它们要么促进要么抑制IRES活性。综上所述，这些结果表明，人类TrkB mRNA的5'前导序列包含多个调控翻译内部起始的顺式元件，并且这种机制可能对神经元树突中TrkB mRNA的翻译有显著贡献。
