Structural and Functional Characterization of Human Telomerase RNA Processing and Cajal Body Localization Signals

Abstract

The RNA component of human telomerase (hTR) includes H/ACA and CR7 domains required for 3' end processing, localization, and accumulation. The terminal loop of the CR7 domain contains the CAB box (ugAG) required for targeting of scaRNAs to Cajal bodies (CB) and an uncharacterized sequence required for accumulation and processing. To dissect out the contributions of the CR7 stem loop to hTR processing and localization, we solved the solution structures of the 3' terminal stem loops of hTR CR7 and U64 H/ACA snoRNA, and the 5' terminal stem loop of U85 C/D-H/ACA scaRNA. These structures, together with analysis of localization, processing, and accumulation of hTRs containing nucleotide substitutions in the CR7 domain, identified the sequence and structural requirements of the hTR processing and CB localization signals and showed that these signals are functionally independent. Further, 3' end processing was found to be a prerequisite for translocation of hTR to CBs.

Dynamics of Human Telomerase Holoenzyme Assembly and Subunit Exchange across the Cell Cycle

“…Combining that observation with the findings presented here, our prediction is that hTERT mRNA translation would prestock newly synthesized hTERT prior to new hTR RNP biogenesis. Because TCAB1 is maximally dissociated from hTR in M and early G 1 phases, and because hTR without TCAB1 and hTERT is nucleolar (15,16,59), hTR synthesized in early G 1 may be trafficked to the nucleoli (Fig. 12).…”

Physiological assembly and activity of human telomerase complexes

“…Thus, the hTR biogenesis pathway is unique among the human H/ACA-motif RNAs (Figure 2A). Notably, if mature hTR is transcribed by RNA Polymerase II within an intron context or if it is transcribed by RNA Polymerase III, only the 3' half of the molecule harboring the H/ACA domain accumulates to detectable level (Theimer et al, 2007;Mitchell et al, 1999a). In addition to its H/ACA motif, hTR also requires a motif termed the biogenesis box (BIO box) for in vivo accumulation (Fu and Collins, 2003).…”

Dyskeratosis congenita mutations in the H/ACA domain of human telomerase RNA affect its assembly into a pre-RNP

“…The consensus sequence of the CAB box is ugAG, where the first two residues are varying and the last two are highly conserved (Richard et al 2003;Jady et al 2004). The CAB box allows Cajal body localization, but it is not involved in 39 end formation of the RNA, indicating that the two processes are functionally independent (Theimer et al 2007). The CR7 also contains key features required for RNP assembly, such as the base pairing between U411 and G417, and an unpaired U418 in the terminal loop.…”

“…One mutation leading to DC is the C408G tranversion, which is located in helix P8b of CR7 (see Fig. 1); this mutation alters the structure of CR7, resulting in the loss of proper processing signals (Theimer et al 2003(Theimer et al , 2007. Another mutation in hTR leading to DC is the deletion of nucleotides 378-451 (D378-451), which removes the entire 39 stem, positioning box H at the 39 extremity.…”