Kickhoefer V A, Liu Y, Kong L B, Snow B E, Stewart P L, Harrington L, Rome L H
Department of Biological Chemistry, University of California at Los Angeles, School of Medicine, Los Angeles, California 90095, USA.
J Cell Biol. 2001 Jan 8;152(1):157-64. doi: 10.1083/jcb.152.1.157.
Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1. Although its role in either complex has not yet been defined, TEP1 has been shown to interact with the mouse telomerase RNA and with several of the human vault RNAs in a yeast three-hybrid assay. An mTep1(-/-) mouse was previously generated which resulted in no apparent change in telomere length or telomerase activity in six generations of mTep1-deficient mice. Here we show that the levels of the telomerase RNA and its association with the telomerase RNP are also unaffected in mTep1(-/-) mice. Although vaults purified from the livers of mTep1(-/-) mice appear structurally intact by both negative stain and cryoelectron microscopy, three-dimensional reconstruction of the mTep1(-/-) vault revealed less density in the cap than previously observed for the intact rat vault. Furthermore, the absence of TEP1 completely disrupted the stable association of the vault RNA with the purified vault particle and also resulted in a decrease in the levels and stability of the vault RNA. Therefore, we have uncovered a novel role for TEP1 in vivo as an integral vault protein important for the stabilization and recruitment of the vault RNA to the vault particle.
穹窿体和端粒酶是含有共同蛋白质亚基TEP1的核糖核蛋白（RNP）颗粒。尽管TEP1在这两种复合体中的作用尚未明确，但在酵母三杂交试验中，已表明TEP1能与小鼠端粒酶RNA以及几种人类穹窿体RNA相互作用。之前培育出了mTep1(-/-)小鼠，在六代mTep1基因缺陷小鼠中，其端粒长度或端粒酶活性均未出现明显变化。在此我们表明，端粒酶RNA的水平及其与端粒酶RNP的结合在mTep1(-/-)小鼠中同样未受影响。尽管通过负染和冷冻电子显微镜观察，从mTep1(-/-)小鼠肝脏中纯化出的穹窿体在结构上看似完整，但对mTep1(-/-)穹窿体的三维重建显示，其帽部的密度低于之前观察到的完整大鼠穹窿体。此外，TEP1的缺失完全破坏了穹窿体RNA与纯化的穹窿体颗粒的稳定结合，还导致穹窿体RNA的水平和稳定性下降。因此，我们发现了TEP1在体内作为一种对穹窿体RNA稳定及募集到穹窿体颗粒至关重要的完整穹窿体蛋白的新作用。
