Georgiev O, Birnstiel M L
EMBO J. 1985 Feb;4(2):481-9. doi: 10.1002/j.1460-2075.1985.tb03654.x.
Analysis of cDNA sequences obtained from the small nuclear RNA U7 has previously suggested specific contacts, by base pairing, between the conserved stem-loop structure and CAAGAAAGA sequence of the histone pre-mRNA and the 5'-terminal sequence of the U7 RNA during RNA processing. In order to test some aspects of the model we have created a series of linker scan, deletion and insertion mutants of the 3' terminus of a sea urchin H3 histone gene and have injected mutant DNAs or in vitro synthesized precursors into frog oocyte nuclei for interpretation. We find that, in addition to the stem-loop structure of the mRNA, the CAAGAAAGA spacer transcript within the histone pre-mRNA is required absolutely for RNA processing, as predicted from our model. Spacer sequences immediately downstream of the CAAGAAAGA motif are not complementary to U7 RNA. Nevertheless, they are necessary for obtaining a maximal rate of RNA processing, as is the ACCA sequence coding for the 3' terminus of the mature mRNA. An increase of distance between the mRNA palindrome and the CAAGAAAGA by as little as six nucleotides abolishes all processing. It may, therefore, be useful to regard both these sequence motifs as part of one and the same RNA processing signal with narrowly defined topologies. Interestingly, U7 RNA-dependent 3' processing of histone pre-mRNA can occur in RNA injection experiments only when the in vitro synthesized pre-mRNA contains sequence extensions well beyond the region of sequence complementarities to the U7 RNA. In addition to directing 3' processing the terminal mRNA sequences may have a role in histone mRNA stabilization in the cytoplasmic compartment.
从小核RNA U7获得的cDNA序列分析先前表明，在RNA加工过程中，组蛋白前体mRNA的保守茎环结构与CAAGAAAGA序列以及U7 RNA的5'-末端序列之间通过碱基配对存在特定的相互作用。为了验证该模型的某些方面，我们构建了一系列海胆H3组蛋白基因3'末端的接头扫描、缺失和插入突变体，并将突变DNA或体外合成的前体注射到蛙卵母细胞核中进行分析。我们发现，正如我们的模型所预测的，除了mRNA的茎环结构外，组蛋白前体mRNA中的CAAGAAAGA间隔转录本对于RNA加工是绝对必需的。CAAGAAAGA基序紧邻下游的间隔序列与U7 RNA不互补。然而，它们对于获得最大的RNA加工速率是必需的，编码成熟mRNA 3'末端的ACCA序列也是如此。mRNA回文序列与CAAGAAAGA之间的距离增加仅6个核苷酸就会消除所有加工。因此，将这两个序列基序视为具有严格定义拓扑结构的同一RNA加工信号的一部分可能是有用的。有趣的是，只有当体外合成的前体mRNA包含远远超出与U7 RNA序列互补区域的序列延伸时，组蛋白前体mRNA的U7 RNA依赖性3'加工才能在RNA注射实验中发生。除了指导3'加工外，末端mRNA序列可能在细胞质区室中组蛋白mRNA的稳定中起作用。
