Biogenesis and assembly of the telomerase RNP

hTERC is transcribed as a precursor and is processed at its 3' end to yield a 451 nucleotide RNA (Zaug et al. 1996). The accumulation of hTERC that has undergone this processing event requires a conserved region of sequence termed the box H/ACA motif (Mitchell et al. 1999a). This motif is bound by a complex containing DKC1 (dyskerin), and mutations in dyskerin affect the processing and accumulation of hTERC (Mitchell et al. 1999b; Mitchell and Collins 2000; Fu and Collins 2003). Studies of purified, catalytically active telomerase indicate that the minimal structure that has telomerase activity in vitro is a complex of one molecule of hTERC RNA, one molecule of hTERT and two molecules of DKC1 (dyskerin) (Cohen et al. 2007). A cryo-electron microscopy (EM) structure of human substrate-bound telomerase holoenzyme revealed that, in addition to one molecule of hTERC RNA, one molecule of hTERT and two molecules of DKC1, the holoenzyme also contains one molecule of WRAP53 (TCAB1, also known as telomere Cajal body protein 1) and two molecules of each NOP10, NHP2 (NOLA2) and GAR1 (Nguyen et al. 2018). WRAP53 is needed for the activity and localization of the telomerase holoenzyme to Cajal bodies (Venteicher et al. 2009). Homozygosity for NHP2 mutations is associated with telomerase failure (dyskeratosis congenita) in humans (Vuillamy et al. 2008). Several additional proteins may associate with the holoenzyme, promoting its assembly and modulating its activity. RUVBL1 (pontin) and RUVBL2 (reptin) are found associated with human telomerase RNPs purified from HeLa cells, and activities of these proteins are required for telomerase RNP assembly in vivo (Venteicher et al. 2008). Pontin and reptin may modulate the interaction between SHQ1 and DKC1 (Machado-Pinilla et al. 2012), but as their exact roles in the assembly and function of telomerase RNP remain unclear, they are annotated simply as positive regulators of telomerase RNP formation.<br><br>The core components hTERC and hTERT undergo trafficking in the cell that may be important for telomerase function. hTERC has been found localized in multiple nuclear structures, including Cajal bodies, nucleoli, and at telomeres (Mitchell et al. 1999a; Jady et al. 2004; Zhu et al. 2004; Jady et al. 2006; Tomlinson et al. 2006). hTERT is also reported localize in Cajal bodies, nucleoli, and to associate with telomeres (Etheridge et al. 2002; Wong et al. 2002; Yang et al. 2002; Zhu et al. 2004; Tomlinson et al. 2006). Some of the factors that regulate trafficking of these two core components of telomerase have been identified, such as nucleolin (Khurts et al. 2004), SMN (Bachand et al. 2002), and 14-3-3 (Seimiya et al. 2000). Cytological studies of HeLa cells suggest that the localization of the telomerase core components can change through the cell-cycle (Jady et al. 2006; Tomlinson et al. 2006). Despite these studies, it is not clear in which compartment hTERT and hTERC assemble to form functional telomerase RNP.<br><br>The assembly of telomerase involves the chaperone proteins p23 and Hsp90, which stably associate with telomerase in vitro (Holt et al. 1999; Forsythe et al. 2001; Keppler et al. 2006). A number of other proteins interact with the telomerase RNP, but it is not clear if they play a role in telomerase assembly. Interestingly, assembled human telomerase RNP can multimerize, though the function of multimerization remains unclear (Beattie et al. 2001; Wenz et al. 2001; Arai et al. 2002).

hTERC作为前体转录物，经其3'端加工后生成含有451个核苷酸的RNA（Zaug等，1996年）。此加工事件产生的hTERC积累需要称为H/ACA盒状结构域的保守序列区域（Mitchell等，1999a）。此结构域被包含DKC1（dyskerin）的复合物所结合，而dyskerin中的突变会影响hTERC的加工和积累（Mitchell等，1999b；Mitchell和Collins，2000；Fu和Collins，2003）。对纯化的、具有催化活性的端粒酶的研究表明，在体外具有端粒酶活性的最小结构是由一分子hTERC RNA、一分子hTERT和两分子DKC1（dyskerin）组成的复合物（Cohen等，2007）。人类底物结合的端粒酶全酶的冷冻电子显微镜（EM）结构揭示，除了含有hTERC RNA一分子、hTERT一分子和DKC1两分子外，全酶还包含WRAP53（TCAB1，亦称为端粒Cajal小体蛋白1）一分子以及NOP10、NHP2（NOLA2）和GAR1各两分子（Nguyen等，2018）。WRAP53对于端粒酶全酶的活性和定位于Cajal小体至关重要（Venteicher等，2009）。NHP2突变纯合子与人类端粒酶功能失败（先天性角化过度症）相关（Vuillamy等，2008）。一些额外的蛋白质可能与全酶结合，促进其组装并调节其活性。RUVBL1（pontin）和RUVBL2（reptin）被发现与从HeLa细胞中纯化的端粒酶RNA复合物相关联，并且这些蛋白质的活性对于端粒酶RNP在体内的组装是必需的（Venteicher等，2008）。Pontin和reptin可能调节SHQ1与DKC1之间的相互作用（Machado-Pinilla等，2012），但由于它们在端粒酶RNP组装和功能中的确切作用尚不明确，它们被注释为端粒酶RNP形成的正调节因子。<br><br>端粒酶的组装涉及伴侣蛋白p23和Hsp90，它们在体外与端粒酶稳定结合（Holt等，1999；Forsythe等，2001；Keppler等，2006）。许多其他蛋白质与端粒酶RNP相互作用，但尚不清楚它们是否在端粒酶组装中发挥作用。有趣的是，组装的人类端粒酶RNP可以多聚化，尽管多聚化的功能尚不清楚（Beattie等，2001；Wenz等，2001；Arai等，2002）。