Distinct Biogenesis Pathways for Human Telomerase RNA and H/ACA Small Nucleolar RNAs  Dragony Fu, Kathleen Collins  Molecular Cell  Volume 11, Issue 5, Pages 1361-1372 (May 2003) DOI: 10.1016/S1097-2765(03)00196-5

Figure 1 Summary of hTR Secondary Structure and Disease-Associated Sequence Changes Dashed boxes indicate the location of deletions or substitutions found in DC or AA patients, with sequence changes in bold. The template/pseudoknot region, H/ACA domain, H/ACA motif region, and CR7 motif are delineated. Molecular Cell 2003 11, 1361-1372DOI: (10.1016/S1097-2765(03)00196-5)

Figure 2 Different Pol II Promoter Types Can Produce Mature and Functional hTR (A) Various promoters were used to drive transcription of the mature hTR sequence with 515 bp of 3′ flanking region. (B) Total RNA prepared from VA13 hTERT cells transiently transfected with empty vector or the constructs in (A) were analyzed by Northern blot hybridization to detect hTR and a total RNA load control (LC). Mature, full-length hTR transcribed in vitro (lane M) or in vivo (lanes 2–4) migrates as a doublet in denaturing PAGE. A linker sequence is present between the CMV transcription initiation site and native hTR sequence, resulting in the slightly slower migration of mature hTR expressed from the CMV promoter. (C) Extracts prepared from the same transfected cells were analyzed for telomerase activity by TRAP. Buffer alone is a negative control for PCR artifacts. The migration of the internal control amplification standard is indicated (IC). Each extract was tested as a series of 5-fold dilutions normalized by total protein. Molecular Cell 2003 11, 1361-1372DOI: (10.1016/S1097-2765(03)00196-5)

Figure 3 Pol II Transcription Termination and 3′ End Processing Elements Inhibit the Production of Mature hTR (A) Genomic sequences downstream of the mature hTR 3′ end in the WT-hTR-500 construct were deleted (WT-hTR-320 or WT-hTR-0) or the entire 515 bp 3′ flanking region was inverted (WT-hTR-α3′). These constructs were transiently transfected into VA13 hTERT cells and total RNA was analyzed by Northern blot hybridization to detect hTR and the load control (LC). M, in vitro transcribed hTR marker. (B) Expression constructs were created to replace hTR 3′ flanking sequence with exogenous Pol II 3′ end processing elements from a typical mRNA (bovine growth hormone polyadenylation [pA] signal) or an snRNA (U1 3′ box/ transcription terminator). (C) Total RNA prepared from VA13 hTERT cells transfected with the constructs indicated in (B) was analyzed by Northern blot hybridization as in (A). The sample in lane 2 ran aberrantly during electrophoresis and does not represent a longer product (note that lanes 2 and 3 are a repeat of lanes 2 and 5 in [A]). (D) The U1 3′ element was shifted further downstream of the hTR 3′ end by ∼160 bp intervals. These constructs were transiently transfected into VA13 hTERT cells and total RNA was analyzed by Northern blot hybridization as in (A). Molecular Cell 2003 11, 1361-1372DOI: (10.1016/S1097-2765(03)00196-5)

Figure 4 The CR7 Motif Is a Telomerase-Specific Substitute for Precursor-Dependent Biogenesis Cues (A) Schematic secondary structures of hTR and the U64, U17, and U67 H/ACA snoRNAs. Dashed boxes expand the hTR CR7 domain and the distal 3′ stem loops of U64, U17, and U67. For stable tetraloop substitutions within hTR or U64, all residues above the line were replaced by a GAAA tetraloop as indicated. (B) The loop of the hTR 3′ stem in the WT-hTR-500 context was substituted with a stable tetraloop to create WT-hTR-500 GAAA. Constructs were transiently transfected into VA13 hTERT cells and total RNA was analyzed by Northern blot hybridization to detect hTR and the load control (LC). M, in vitro transcribed full-length hTR marker. (C) The hTR H/ACA domain (nt 211-451), the H/ACA domain with a 3′ loop tetraloop substitution, and the H/ACA domain with the ACA motif changed to TGT were expressed in native hTR context. Total RNA from transiently transfected VA13 hTERT cells was analyzed by Northern blot hybridization with an oligonucleotide complementary to the hTR H/ACA domain; the standard load control (LC) was detected in a subsequent hybridization. M, in vitro transcribed hTR H/ACA domain marker. (D) U64 H/ACA snoRNA was expressed from the CMV promoter with hTR 3′ genomic downstream flanking sequence, with or without the terminal 3′ loop tetraloop substitution. These constructs were transiently transfected into 293 cells and total RNA was analyzed by Northern blot hybridization with an oligonucleotide complementary to U64. Note that 293 cells contain endogenous U64 at a level evident in cells transfected with vector alone (lane 1) or with the expression construct encoding an altered mobility U64 (lane 3). Expression of a cotransfected plasmid control RNA was also detected (LC). M, in vitro transcribed U64 marker extended by the length of the cloning polylinker. (E) The H/ACA domain of hTR within WT-hTR-500 was substituted with mature U64, U17, or U67 (left) or U64 with 3′ loop tetraloop substitution (right). Total RNA from 293 cells transfected with these constructs was analyzed by Northern blot hybridization with an oligonucleotide that detects endogenous full-length hTR, all chimeric RNAs, and the standard load control (LC). Asterisks denote crossreacting RNAs detected in 293 cells transfected with vector alone (not shown). M, in vitro transcribed full-length hTR marker. Molecular Cell 2003 11, 1361-1372DOI: (10.1016/S1097-2765(03)00196-5)

Figure 5 DC and AA hTR Variants Are Defective in Cellular Accumulation and/or Enzyme Activity (A) Total RNA from VA13 hTERT cells transfected with WT-hTR-500 constructs containing DC or AA associated hTR mutations was analyzed by Northern blot hybridization. hTR accumulation was quantified by phosphorimager, normalized according to total RNA loading and transfection controls (not shown), and expressed relative to wild-type as “relative accumulation.” (B) Telomerase activity from extracts of the same cells analyzed in (A) was examined by TRAP. Each sample was tested in a series of 5-fold extract dilutions normalized by total protein. Relative activity was scored as the number of titration points that yielded an activity assay ladder. The migration of the internal control amplification standard is indicated (IC). (C) Telomerase RNP was reconstituted with recombinant hTERT and the indicated hTR variant in 5-fold steps of RNA concentration, from 5–625 nM in the reconstitution reaction. Telomerase activity was detected by TRAP. Relative activity was scored as in (B). The migration of the internal control amplification standard is indicated (IC). Molecular Cell 2003 11, 1361-1372DOI: (10.1016/S1097-2765(03)00196-5)

Figure 6 Comparison of Biogenesis Pathways for snoRNAs and hTR Double-headed arrows represent interactions within the processing cascade. See key and text for additional details. Molecular Cell 2003 11, 1361-1372DOI: (10.1016/S1097-2765(03)00196-5)