La相关蛋白7(LARP7)是保护RNA3'-末端的RNA伴侣家族,是特定核糖核蛋白复合物(RNP)的组分。嗜热四膜虫端粒酶,LARP7蛋白p65与端粒酶逆转录酶(TERT)和端粒酶RNA(TER)一起形成核心RNP。p65有四个已知的结构域-N端结构域(NTD),La主题(LaM),RNA识别基序1(RRM1),和C端xRRM2。迄今为止,只有xRRM2和LaM以及它们与TER的相互作用在结构上进行了表征。导致低温EM密度图分辨率低的构象动力学限制了我们对全长p65如何特异性识别和重塑TER以进行端粒酶组装的理解。这里,我们将四膜虫端粒酶冷冻EM图的集中分类与NMR光谱相结合,以确定p65-TER的结构。确定了三个以前未知的螺旋,在绑定La模块的本质上无序的NTD中,一个扩展RRM1,另一个在xRRM2之前,稳定p65-TER相互作用。扩展的La模块(αN,LaM和RRM1)与四个3'末端U核苷酸相互作用,而LaM和αN另外与TER伪结相互作用,和LaM与茎1和5'端。我们的结果表明,广泛的p65-TER相互作用促进了TER3'端保护,TER折叠,和核心RNP组装和稳定。带有TER的全长p65的结构也揭示了真正的La和LARP7蛋白作为RNA伴侣和核心RNP成分的生物学作用。
La-related protein 7 (LARP7) are a family of RNA chaperones that protect the 3\'-end of RNA and are components of specific ribonucleoprotein complexes (RNP). In Tetrahymena thermophila telomerase, LARP7 protein p65 together with telomerase reverse transcriptase (TERT) and telomerase RNA (TER) form the core RNP. p65 has four known domains-N-terminal domain (NTD), La motif (LaM), RNA recognition motif 1 (RRM1), and C-terminal xRRM2. To date, only the xRRM2 and LaM and their interactions with TER have been structurally characterized. Conformational dynamics leading to low resolution in cryo-EM density maps have limited our understanding of how full-length p65 specifically recognizes and remodels TER for telomerase assembly. Here, we combined focused classification of Tetrahymena telomerase cryo-EM maps with NMR spectroscopy to determine the structure of p65-TER. Three previously unknown helices are identified, one in the otherwise intrinsically disordered NTD that binds the La module, one that extends RRM1, and another preceding xRRM2, that stabilize p65-TER interactions. The extended La module (αN, LaM and RRM1) interacts with the four 3\' terminal U nucleotides, while LaM and αN additionally interact with TER
pseudoknot, and LaM with stem 1 and 5\' end. Our results reveal the extensive p65-TER interactions that promote TER 3\'-end protection, TER folding, and core RNP assembly and stabilization. The structure of full-length p65 with TER also sheds light on the biological roles of genuine La and LARP7 proteins as RNA chaperones and core RNP components.