Harding Heather P, Lackey Jeremy G, Hsu Hao-Chi, Zhang Yuhong, Deng Jing, Xu Rui-Ming, Damha Masad J, Ron David
Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, New York 10016, USA.
RNA. 2008 Feb;14(2):225-32. doi: 10.1261/rna.859908. Epub 2007 Dec 19.
Unconventional mRNA splicing by an endoplasmic reticulum stress-inducible endoribonuclease, IRE1, is conserved in all known eukaryotes. It controls the expression of a transcription factor, Hac1p/XBP-1, that regulates gene expression in the unfolded protein response. In yeast, the RNA fragments generated by Ire1p are ligated by tRNA ligase (Trl1p) in a process that leaves a 2'-PO4(2-) at the splice junction, which is subsequently removed by an essential 2'-phosphotransferase, Tpt1p. However, animals, unlike yeast, have two RNA ligation/repair pathways that could potentially rejoin the cleaved Xbp-1 mRNA fragments. We report that inactivation of the Trpt1 gene, encoding the only known mammalian homolog of Tpt1p, eliminates all detectable 2'-phosphotransferase activity from cultured mouse cells but has no measurable effect on spliced Xbp-1 translation. Furthermore, the relative translation rates of tyrosine-rich proteins is unaffected by the Trpt1 genotype, suggesting that the pool of (normally spliced) tRNA(Tyr) is fully functional in the Trpt1-/- mouse cells. These observations argue against the presence of a 2'-PO4(2-) at the splice junction of ligated RNA molecules in Trpt1-/- cells, and suggest that Xbp-1 and tRNA ligation proceed by distinct pathways in yeast and mammals.
内质网应激诱导的核糖核酸内切酶IRE1介导的非常规mRNA剪接在所有已知真核生物中都保守存在。它控制转录因子Hac1p/XBP-1的表达，该转录因子在未折叠蛋白反应中调节基因表达。在酵母中，Ire1p产生的RNA片段由tRNA连接酶（Trl1p）连接，在此过程中，剪接连接处会留下一个2'-磷酸（2-），随后由一种必需的2'-磷酸转移酶Tpt1p将其去除。然而，与酵母不同的是，动物有两条RNA连接/修复途径，可能会重新连接切割后的Xbp-1 mRNA片段。我们报道，编码Tpt1p唯一已知哺乳动物同源物的Trpt1基因失活，消除了培养的小鼠细胞中所有可检测到的2'-磷酸转移酶活性，但对剪接后的Xbp-1翻译没有可测量的影响。此外，富含酪氨酸的蛋白质的相对翻译速率不受Trpt1基因型的影响，这表明（正常剪接的）tRNA（Tyr）库在Trpt1基因敲除小鼠细胞中功能完全正常。这些观察结果表明，在Trpt1基因敲除细胞中，连接的RNA分子的剪接连接处不存在2'-磷酸（2-），并表明Xbp-1和tRNA连接在酵母和哺乳动物中通过不同的途径进行。
