Reconstitution and characterization of the unconventional splicing of XBP1u mRNA in vitro.
Reconstitution and characterization of the unconventional splicing of XBP1u mRNA in vitro.
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DOI：
10.1093/nar/gkr132
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发表时间：
2011-07
影响因子：
14.9
通讯作者：
Kohno K
中科院分区：
文献类型：
--
作者：
Shinya S;Kadokura H;Imagawa Y;Inoue M;Yanagitani K;Kohno K
在内质网（ER）应激时，哺乳动物细胞诱导转录激活因子XBP 1 s的合成以缓解应激。在非应激条件下，XBP 1 s的信使RNA（mRNA）以未剪接的前体形式存在于胞质溶胶中，即XBP 1u mRNA。因此，必须去除其内含子以合成XBP 1。在ER应激时，应激传感器IRE 1 α切割XBP 1u mRNA，以启动ER膜上XBP 1u mRNA的非常规剪接。释放的两个外显子连接形成成熟的XBP 1 s mRNA。然而，这种剪接的机制仍然不清楚，主要是因为连接XBP 1 s mRNA一半的酶是未知的。在这里，我们重建了体外的整个剪接反应的XBP 1u mRNA。使用该测定，我们表明，与体内研究一致，哺乳动物胞质溶胶确实具有可以连接XBP 1 s mRNA一半的RNA连接酶。IRE 1 α对XBP 1u mRNA的切割在切割位点产生了2′，3′-环状磷酸结构。有趣的是，这种磷酸盐被胞质溶胶中的酶掺入到XBP 1 s mRNA中，以连接两个外显子。这些研究揭示了测定系统的实用性和哺乳动物胞质酶的独特性质，可以促进XBP 1u mRNA的剪接。
Upon endoplasmic reticulum (ER) stress, mammalian cells induce the synthesis of a transcriptional activator XBP1s to alleviate the stress. Under unstressed conditions, the messenger RNA (mRNA) for XBP1s exists in the cytosol as an unspliced precursor form, XBP1u mRNA. Thus, its intron must be removed for the synthesis of XBP1s. Upon ER stress, a stress sensor IRE1α cleaves XBP1u mRNA to initiate the unconventional splicing of XBP1u mRNA on the ER membrane. The liberated two exons are ligated to form the mature XBP1s mRNA. However, the mechanism of this splicing is still obscure mainly because the enzyme that joins XBP1s mRNA halves is unknown. Here, we reconstituted the whole splicing reaction of XBP1u mRNA in vitro. Using this assay, we showed that, consistent with the in vivo studies, mammalian cytosol indeed had RNA ligase that could join XBP1s mRNA halves. Further, the cleavage of XBP1u mRNA with IRE1α generated 2′, 3′-cyclic phosphate structure at the cleavage site. Interestingly, this phosphate was incorporated into XBP1s mRNA by the enzyme in the cytosol to ligate the two exons. These studies reveal the utility of the assay system and the unique properties of the mammalian cytosolic enzyme that can promote the splicing of XBP1u mRNA.
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影响因子：
64.5
作者：
Yoshida, H;Matsui, T;Mori, K
通讯作者：
Mori, K
DOI：
10.1073/pnas.82.3.684
发表时间：
1985-01-01
影响因子：
11.1
作者：
PERKINS, KK;FURNEAUX, H;HURWITZ, J
通讯作者：
HURWITZ, J
影响因子：
5.3
作者：
ZILLMANN, M;GOROVSKY, MA;PHIZICKY, EM
通讯作者：
PHIZICKY, EM
影响因子：
82.9
作者：
Iwawaki, T;Akai, R;Miura, M
通讯作者：
Miura, M
影响因子：
64.5
作者：
Sidrauski, C;Walter, P
通讯作者：
Walter, P