Multiple requirements for nematode spliced leader RNP function in trans-splicing.
Multiple requirements for nematode spliced leader RNP function in trans-splicing.
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发表时间：
1996-08
期刊：
影响因子：
4.5
通讯作者：
J. Denker;P. Maroney;Yi-Tao Yu;R. A. Kanost;Timothy W. Nilsen
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作者：
J. Denker;P. Maroney;Yi-Tao Yu;R. A. Kanost;Timothy W. Nilsen
线虫转拼中的 5' 外显子供体 SL RNA 是一种小型（约 100 nt）RNA，类似于顺式剪接体 U snRNA。对 SL RNA 功能所需 RNA 序列的广泛分析揭示了四个必需元件：核心 Sm 结合位点、紧邻该位点下游的三个核苷酸、干环 II 区域和 5' 剪接位点。尽管这些元件对于 SL RNA 的体外功能是必要且充分的，但它们各自在促进 SL RNA 活性中的作用尚未阐明。此外，虽然已表明SL RNA组装成Sm RNP是功能的先决条件，但SL RNP的蛋白质组成尚未确定。在这里，我们使用寡核糖核苷酸亲和力来纯化 SL RNP，发现它含有核心 Sm 蛋白以及四种特定蛋白（175、40、30 和 28 kDa）。使用体外组装测定；我们发现 175-kDa 和 30-kDa SL 特异性蛋白的关联与反式剪接中的 SL RNP 功能相关。这些蛋白质的结合取决于核心 Sm 结合位点的序列；含有 U1 snRNA Sm 结合位点的 SL RNA 组装成含有核心蛋白但不含有 SL 特异性蛋白的 Sm RNP。此外，突变和硫代磷酸盐干扰方法揭示了 SL RNA 的 Stemloop II 片段内的一级核苷酸序列和特定磷酸氧都是功能所必需的。最后，SL 序列本身内一个不寻常的隐秘 5' 剪接位点的突变激活表明 U5 snRNA 可能在选择和指定 SL 添加反式剪接中的 5' 剪接位点方面发挥主要作用。
The 5' exon donor in nematode trans-splicing, the SL RNA, is a small (approximately 100 nt) RNA that resembles cis-spliceosomal U snRNAs. Extensive analyses of the RNA sequence requirements for SL RNA function have revealed four essential elements, the core Sm binding site, three nucleotides immediately downstream of this site, a region of Stem-loop II, and a 5' splice site. Although these elements are necessary and sufficient for SL RNA function in vitro, their respective roles in promoting SL RNA activity have not been elucidated. Furthermore, although it has been shown that assembly of the SL RNA into an Sm RNP is a prerequisite for function, the protein composition of the SL RNP has not been determined. Here, we have used oligoribonucleotide affinity to purify the SL RNP and find that it contains core Sm proteins as well as four specific proteins (175, 40, 30, and 28 kDa). Using in vitro assembly assays; we show that association of the 175- and 30-kDa SL-specific proteins correlates with SL RNP function in trans-splicing. Binding of these proteins depends upon the sequence of the core Sm binding site; SL RNAs containing the U1 snRNA Sm binding site assemble into Sm RNPs that contain core, but not SL-specific proteins. Furthermore, mutational and thiophosphate interference approaches reveal that both the primary nucleotide sequence and a specific phosphate oxygen within a segment of Stemloop II of the SL RNA are required for function. Finally, mutational activation of an unusual cryptic 5' splice site within the SL sequence itself suggests that U5 snRNA may play a primary role in selecting and specifying the 5' splice site in SL addition trans-splicing.