INVITRO ASSEMBLY OF YEAST U6 SNRNP - A FUNCTIONAL ASSAY
INVITRO ASSEMBLY OF YEAST U6 SNRNP - A FUNCTIONAL ASSAY
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DOI：
10.1101/gad.3.12b.2137
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发表时间：
1989-12-01
影响因子：
10.5
通讯作者：
ABELSON, J
中科院分区：
文献类型：
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作者：
FABRIZIO, P;MCPHEETERS, DS;ABELSON, J
U6 小核 RNA (snRNA) 是最高度保守的剪接体 RNA，人们推测它在前 mRNA 剪接中具有重要作用。为了阐明这一作用，我们开发了一种体外系统来重建功能性 U6 小核糖核蛋白 (snRNP)。用与 U6 snRNA 中心结构域互补的寡核苷酸处理剪接提取物会导致 RNase H 切割内源 U6 snRNA 并丧失剪接活性。然后将使用 T7 RNA 聚合酶在体外合成的酵母 U6 RNA 添加到寡核苷酸处理的提取物中，并通过随后添加底物前 mRNA 来监测剪接活性的恢复。将全长、未修饰的 T7U6 snRNA（113 个核苷酸）添加到寡核苷酸处理的提取物中可有效恢复剪接活性。使用在 3'' 末端截短的 U6 RNA 转录本，我们发现大的缺失（39 个核苷酸）产生的分子无法在体外恢复剪接活性，也不能与内源性 U4 snRNA 相互作用或形成成熟的剪接体。最后，我们证明，T7U6 RNA 中不变的 G81 被 C 取代会消除其恢复剪接活性的能力。尽管 U4/U6 snRNP 正确形成，但成熟的剪接体无法组装。
U6 small nuclear RNA (snRNA) is the most highly conserved spliceosomal RNA, and it has been postulated to have a fundamental role in pre-mRNA splicing. To elucidate this role, we developed an in vitro system for reconstituting the functional U6 small ribonucleoprotein (snRNP). Treating splicing extracts with an oligonucleotide complementary to the central domain of U6 snRNA leads to both RNase H cleavage of the endogenous U6 snRNA and loss of splicing activity. Yeast U6 RNA, synthesized in vitro using T7 RNA polymerase, is then added to the oligonucleotide-treated extract, and restoration of splicing activity is monitored by the subsequent addition of substrate pre-mRNA. Addition of full-length, unmodified T7U6 snRNA (113 nucleotides) to oligonucleotide-treated extracts restores splicing activity efficiently. Using U6 RNA transcripts truncated at their 3'' ends, we show that large deletions (39 nucleotides) produce molecules that are unable to restore splicing activity in vitro and cannot interact with the endogenous U4 snRNA or form a mature spliceosome. Finally, we show that substitution of the invariant G81 with C within the T7U6 RNA abolishes its ability of restoring splicing activity. Although the U4/U6 snRNP forms correctly, mature spliceosomes do not assemble.