Department of Biology, York University, 4700 Keele St., Life Science Building #327E, Toronto, ON M3J 1P3, Canada.
Nucleic Acids Res. 2018 May 4;46(8):4228-4240. doi: 10.1093/nar/gky090.
In addition to a role in the processing of nascent RNA polymerase III transcripts, La proteins are also associated with promoting cap-independent translation from the internal ribosome entry sites of numerous cellular and viral coding RNAs. La binding to RNA polymerase III transcripts via their common UUU-3'OH motif is well characterized, but the mechanism of La binding to coding RNAs is poorly understood. Using electromobility shift assays and cross-linking immunoprecipitation, we show that in addition to a sequence specific UUU-3'OH binding mode, human La exhibits a sequence specific and length dependent poly(A) binding mode. We demonstrate that this poly(A) binding mode uses the canonical nucleic acid interaction winged helix face of the eponymous La motif, previously shown to be vacant during uridylate binding. We also show that cytoplasmic, but not nuclear La, engages poly(A) RNA in human cells, that La entry into polysomes utilizes the poly(A) binding mode, and that La promotion of translation from the cyclin D1 internal ribosome entry site occurs in competition with cytoplasmic poly(A) binding protein (PABP). Our data are consistent with human La functioning in translation through contacts to the poly(A) tail.
除了在新生 RNA 聚合酶 III 转录本的加工中发挥作用外，La 蛋白还与促进许多细胞和病毒编码 RNA 的内部核糖体进入位点的无帽依赖性翻译有关。La 通过其共同的 UUU-3'OH 基序与 RNA 聚合酶 III 转录本结合的机制已经得到很好的描述，但 La 与编码 RNA 结合的机制尚不清楚。我们使用电泳迁移率变动分析和交联免疫沉淀实验表明，除了具有序列特异性的 UUU-3'OH 结合模式外，人 La 还表现出序列特异性和长度依赖性多聚 A 结合模式。我们证明，这种多聚 A 结合模式使用了先前在尿嘧啶结合过程中被证明为空的命名为 La 基序的典型核酸相互作用翼状螺旋面。我们还表明，细胞质而非核 La 在人细胞中与多聚 A RNA 结合，La 进入多核糖体利用多聚 A 结合模式，并且 La 促进细胞周期蛋白 D1 内部核糖体进入位点的翻译与细胞质多聚 A 结合蛋白 (PABP) 竞争。我们的数据表明，人 La 通过与多聚 A 尾巴的接触在翻译中发挥作用。
