Harris K A, Crothers D M, Ullu E
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA.
RNA. 1995 Jun;1(4):351-62.
The formation of the mRNA 5' end in trypanosomatid protozoa is carried out by trans-splicing, which transfers a spliced leader (SL) sequence and its hypermethylated cap (cap4) from the SL RNA to the pre-mRNA. Previous in vitro studies with synthetic uncapped RNAs have shown that the SL sequence of Leptomonas collosoma can assume two alternate conformations, Form 1 and Form 2, with Form 1 being the dominant one. To gain information about the structure of the SL RNA in vivo, in its protein-rich environment, we have used permeable Trypanosoma brucei and L. collosoma cells for chemical modification experiments. We introduce the use in vivo of the water-soluble reagents CMCT and kethoxal. In contrast to the in vitro results, the Form 2 secondary structure predominates. However, there are chemically accessible regions that suggest conformational flexibility in SL RNPs and a chemically inaccessible region suggestive of protection by protein or involvement in tertiary interactions. Using complementary 2'-O-methyl RNA oligonucleotides, we show that T. brucei SL RNA can be induced to switch conformation in vivo. SL RNA stripped of proteins and probed in vitro does not display the same Form 2 bias, indicating that SL RNA structure is determined, at least in part, by its RNP context. Finally, the methyl groups of the cap4 do not seem to affect the secondary structure of T. brucei SL RNA, as shown by chemical modification of undermethylated SL RNA probed in vivo.
锥虫原生动物中mRNA 5'端的形成是通过反式剪接完成的，反式剪接将一个剪接前导序列（SL）及其超甲基化帽（cap4）从SL RNA转移到前体mRNA上。先前对合成的无帽RNA进行的体外研究表明，粗线椎虫的SL序列可以呈现两种交替构象，即形式1和形式2，其中形式1占主导。为了获取关于体内富含蛋白质环境中SL RNA结构的信息，我们使用了可渗透的布氏锥虫和粗线椎虫细胞进行化学修饰实验。我们介绍了水溶性试剂CMCT和乙二醛在体内的应用。与体外结果相反，形式2的二级结构占主导。然而，存在化学可及区域，这表明SL核糖核蛋白颗粒（RNP）具有构象灵活性，还有一个化学不可及区域，提示受到蛋白质保护或参与三级相互作用。使用互补的2'-O-甲基RNA寡核苷酸，我们表明布氏锥虫SL RNA在体内可被诱导发生构象转换。去除蛋白质并在体外进行探测的SL RNA并未表现出相同的形式2偏向性，这表明SL RNA的结构至少部分由其RNP环境决定。最后，体内探测的低甲基化SL RNA的化学修饰表明，cap4的甲基似乎不影响布氏锥虫SL RNA的二级结构。
