Spliced leader RNA of trypanosomes: In vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation
Spliced leader RNA of trypanosomes: In vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation
复制标题
DOI：
10.1002/j.1460-2075.1996.tb00811.x
复制
发表时间：
1996-08-15
期刊：
影响因子：
11.4
通讯作者：
Bindereif, A
中科院分区：
文献类型：
--
作者：
Lucke, S;Xu, GL;Bindereif, A
在锥虫体中，mRNAs是通过反式剪接产生的，剪接的前导(SLRNA)RNA在反式剪接过程中起着核心作用，它向每个蛋白质编码外显子提供5‘端的微型外显子。我们建立了III活体实验来研究锥虫物种西莫里锥虫SL RNA的剪接、Cap4修饰和RNP组装。首先，我发现在体内SL RNA的功能需要微小外显子内的大量序列，尽管保守长度39个核苷酸并不是必需的，相反，内含子序列似乎对突变具有令人惊讶的耐受性；只有茎环LI结构是必不可少的，茎I区序列要求的不对称性表明这个结构域可能存在于不同的功能构象中。第二，修饰位点外的不同的微小外显子序列对于有效的Cap4形成是重要的。第三，所有测试的SL RNA突变都允许核心RNP组装，这表明对核心蛋白结合的灵活要求。总之，我们的突变分析结果为SL RNA的离散结构域结构提供了证据，并有助于解释微小外显子序列和整个SL RNA二级结构的强大系统发育保守性；它们还表明线虫和锥虫中的反式剪接之间可能存在某些差异。该方法为研究反式剪接体中的RNA-RNA相互作用提供了基础。
In trypanosomes mRNAs are generated through trans splicing, The spliced leader (SL) RNA, which donates the 5'-terminal mini-exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established iii vivo assays to study iu detail ti ans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, me found that extensive sequences within the mini-exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential, In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stemloop LI structure is indispensable, The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations, Second, distinct mini-exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding, In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini-exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA-RNA interactions in the trans spliceosome.