La protein has a positive effect on the translation of TOP mRNAs in vivo

In vertebrates, the mRNAs encoding ribosomal proteins, as well as other proteins implicated in translation, are characterized by a 5′-untranslated region (5′-UTR), including a stretch of pyrimidines at the 5′-end. The 5′-terminal oligopyrimidine (5′-TOP) sequence, which is involved in the growth-dependent translational regulation characteristic of this class of genes (so-called TOP genes), has been shown to specifically bind the La protein in vitro, suggesting that La might be implicated in translational regulation in vivo. In order to substantiate this hypothesis, we have examined the effect of La on TOP mRNA translational control in both stable and transient transfection experiments. In particular we have constructed and analyzed three stably transfected Xenopus cell lines inducible for overexpression of wild-type La or of putative dominant negative mutated forms. Moreover, La-expressing plasmids have been transiently co-transfected together with a plasmid expressing a reporter TOP mRNA in a human cell line. Our results suggest that in vivo La protein plays a positive role in the translation of TOP mRNA. They also suggest that the function of La is to counteract translational repression exerted by a negative factor, possibly cellular nucleic acid binding protein (CNBP), which has been previously shown to bind the 5′-UTR downstream from the 5′-TOP sequence.

在脊椎动物中，编码核糖体蛋白以及其他翻译相关蛋白的mRNA，均带有5'非翻译区（5′-UTR），该区域的5'端包含一段嘧啶序列。5'末端寡嘧啶序列（5′-TOP）参与这类基因（即所谓的TOP基因）所特有的生长依赖性翻译调控过程；体外实验已证实该序列可特异性结合La蛋白，这提示La蛋白可能在体内参与翻译调控。为验证这一假说，本研究在稳定转染与瞬时转染两类实验中，均考察了La蛋白对TOP mRNA翻译调控的影响。具体而言，本研究构建并分析了三种可诱导过表达野生型La蛋白或推定显性负突变体的稳定转染爪蟾细胞系。此外，本研究还在人细胞系中，将携带La蛋白编码基因的质粒与表达报告型TOP mRNA的质粒共同进行瞬时转染。本研究结果表明，La蛋白在体内可对TOP mRNA的翻译发挥正向调控作用；同时提示，La蛋白的功能是拮抗某一负性因子所介导的翻译抑制，该负性因子可能为细胞核酸结合蛋白（CNBP）——此前已有研究证实其可结合5′-TOP序列下游的5'非翻译区。