1. Volume 27, Issue 6, Pages 869-881 (September 2007)
Structural and Functional Characterization of Human Telomerase RNA Processing and Cajal Body Localization Signals 
Carla A. Theimer, Beáta E. Jády, Nicholas Chim, Patricia Richard, Katherine E. Breece, Tamás Kiss, Juli Feigon 
Molecular Cell 
Volume 27, Issue 6, Pages (September 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
Secondary Structures of Human U64 H/ACA snoRNA, hTR, and U85 C/D-H/ACA scaRNA
(A) Schematic of full-length RNAs. The loop sequences investigated in this study are indicated by boxes.
(B) Sequences of the U64t, CR7t, and U85t NMR constructs. Residues are numbered to match the full-length RNA sequences. Boxed sequences on U85t and CR7t indicate the four nts in the CB localization signal. Nts below the dotted line are non-native sequences added during construct design.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2
Structures of Terminal Loops of U64 H/ACA snoRNA (3′ Hairpin), hTR CR7, and U85 C/D-H/ACA scaRNA (5′ Hairpin)
Solution structures of (A–C) U64t, (D–F) CR7t, and (G–I) U85t.
(A, D, and G) Superpositions of the 20 lowest energy structures.
(B, E, and H) Stereoviews of the lowest energy structure of the loops (minor groove view). The gray ribbon through the phosphate atoms (yellow spheres) is used to indicate the backbone topology.
(C, F, and I) Schematic representations of the loops. Nts are colored A (orange), G (blue), U (green) and C (red).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
CR7 Loop-Dependent Processing and Intranuclear Localization of hTR
(A) Schematic of hTR expression construct. The HTR gene (open arrow) with its 449 bp upstream and 66 bp downstream sequences was PCR amplified and inserted into the XhoI (X) and BamHI (B) sites of pBluescript. The multiple cloning site (MCS) and T7 promoter are indicated. The RNA probe used for RNase A/T1 protection is shown. Positions of the fluorescently labeled oligont FISH probes specific for hTR (red) or pre-hTR (green) are shown.
(B) RNase A/T1 mapping. Transient expression of hTRs was verified both in VA13 cells that contain no endogenous hTR and in HeLa cells (data not shown). RNAs extracted from VA13 cells either transfected (T) or nontransfected (N) with the pHTR (WT) or pHTR-Lcomp expression plasmids were hybridized with internally labeled complementary RNA probe, digested with RNase A and T1, and fractionated on a 4% sequencing acrylamide gel. RNAs representing mature (closed arrow) and precursor hTRs (open arrow with 3′ tail) are indicated. Please note that mature hTR and its protected complementary RNA migrate aberrantly. Lane C, control mapping with E. coli tRNA. Lane M, molecular markers (terminally labeled HaeIII- and TaqI-digested pBR322).
(C) In situ localization of hTRs. Transiently expressed hTRs carrying WT or altered sequences were localized in HeLa cells. Accumulation of mature (closed arrow) or precursor hTRs (open arrow) was verified by RNase A/T1 mapping both in HeLa and VA13 cells. Cells were hybridized with fluorescently labeled oligonts complementary to hTR from A346 to A382 or to the 3′ flanking sequences of hTR from U+27 to U+55. Nu and CBs were visualized by expression of fibrillarin-GFP and coilin-GFP, respectively. Scale bars, 10 μm.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Transient Expression and Localization of the 3′ Terminal H/ACA Domain of hTR
(A) Schematic structure of the pCMV-globin-hTR-3′ expression construct. The exons (E1, E2, and E3) and the polyadenylation site (PA) of the human β-globin gene and the promoter region of the cytomegalovirus (CMV) are shown. The 3′ terminal H/ACA domain of hTR is represented by a broken arrow. Relevant restriction sites are shown (H, HindIII; C, ClaI, X, XhoI).
(B) RNase A/T1 mapping. RNAs obtained from HeLa cells either transfected (T) or nontransfected (N) with the pCMV-globin expression vector carrying the 3′ terminal H/ACA domains of the WT (WT) or mutant (CABcomp) hTRs were analyzed by RNase A/T1 mapping. The structure and length of hTRs protecting RNA probes are shown on the right. Asterisks indicate hTR-3′ RNA processed at C221. The 451 and 410 nt fragments represent fully and partially protected endogenous hTR, respectively. Lanes C, control mappings. Lane M, size markers.
(C) In situ localization of hTRs processed from the second intron of transiently expressed globin pre-mRNA. For details, see the legend to Figure 3C.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
CR7 Stem P8b-Dependent Processing and Intranuclear Localization of hTR
(A) Transient expression and in situ localization of hTR containing the DKC mutation C408G and P8b stem base pair substitutions. For details, see legend to Figure 3C.
(B) Secondary structures of the hTR CR7 domain C408G and WT. C408G rearranges the phylogenetically conserved base pairing of the CR7 P8b stem (blue nts) (Theimer et al., 2003). The C408G and C408G/G421C compensatory mutations are circled in red.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Schematic Representations of the hTR CR7 Hairpin Loop
(A) The hTR-specific processing signal. (B) The hTR CB localization signal. (C) Phylogenetic conservation among vertebrate species in the Rfam database (Griffiths-Jones et al., 2003). Red boxes in (A) indicate nts that have a significant effect on correct hTR processing. Dark blue, light blue, and white boxes in (B) indicate the four CAB box nts, nts that attenuate localization, and nts that had no effect on CB localization, respectively. Dark green, light green, and white boxes in (C) indicate >95% conservation, >85%–95% conservation, and ≤85% conservation, respectively. Conserved bps are indicated by dark green lines.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions