Article Figures & Data
Figures
- Fig. 1.
Potential secondary structures of the 3′ ends of hTR and mTR. The structures (hTR nt 211 to 451 and mTR nt 169 to 397 as indicated) are based on the conserved hairpin-hinge-hairpin-tail secondary structure of box H/ACA snoRNAs and a sequence comparison between hTR and mTR. H and ACA box primary sequence elements are contained within the light and dark boxes, respectively.
- Fig. 2.
hTR expression by Pol II. (A) Schematic of hTR Pol II expression system indicates relative positions of restriction endonuclease sites, template residues, H and ACA box residues, sequence tag insertion site (StuI), and probes used in Northern blot analyses (h1 to h6, αTag). Processed hTR species are indicated by gray bars and correspond according to the number of filled circles with those in panel B. (B and C) Northern blot analysis of total RNA prepared from transiently transfected 293 cells probed with αTag oligonucleotide (B) or stripped and reprobed with h4 and h5 oligonucleotides (C). Lane 1, mock transfection; lane 2, empty pRc/CMV vector; lanes 3 to 11, hTR restriction fragments cloned into pRc/CMVHindIII site (except lane 4 [into NotI site]). Restriction fragments: lanes 3 and 4, SE; lane 5, XE (note for panel C that construct does not contain the sequences complementary to h4 or h5); lane 6, SN; lane 7, SA; lanes 8 to 11, SE with the following mutations in the ACA and H boxes: ACA→TCA,ACA→TGT, AGAGGA→ATATTA, andAGAGGA→TGTGGT, respectively (mutated residues are underlined). Filled circles indicate processed hTR species in lane 3. Lane i contains in vitro-transcribed hTR standards: E and N are transcripts from the sequence-tagged SE fragment in pRc/CMV driven by the T7 promoter and terminated approximately at the E and N restriction sites, respectively. A 110-nt RNA RC was included during sample preparation to verify loading equivalents. In lanes 2 to 11, the U6tTR construct was included as a control for relative transfection efficiency (TC). Cross-reacting endogenous non-hTR RNAs are indicated by an asterisk. (D) Northern blot analysis performed as described above and probed with αTag oligonucleotide. Lane 12, SE; lane 13, SN with restored ACA box. Note that both endogenous and recombinant hTR, as well as hTR transcribed in vitro by T7 RNA polymerase, migrate as doublets in a denaturing polyacrylamide gel.
- Fig. 3.
Recombinant hTR incorporation into an active telomerase RNP. (A) Northern blot analysis of total RNA prepared from cells transiently transfected with WT and AT versions of the sequence-tagged SE construct (arrowheads). The blot was probed with αTag oligonucleotide. Lane i contains in vitro-transcribed hTR standards E and N. (B) TRAP activity assay of extracts prepared from WT and AT transiently transfected 293 cells. Oligonucleotides amplifying WT (C3TA2) or AT (A2C4, C4A2, C2A2C2) telomerase extension products were used as indicated. For lanes 9 to 12 and 21 to 24, extracts were incubated with RNase A prior to the telomerase extension reaction. Each pair of lanes corresponds to 20- and 100-fold dilutions of whole-cell extracts.
- Fig. 4.
Processing, accumulation, and activity of a chimeric hTR-U64 RNA. (A) Northern blot analysis of total RNA prepared from cells transiently transfected with the following constructs: sequence-tagged AT SE (solid arrowheads), AT hTR-U64, and WT hTR-U64 (open arrowhead). The blot was probed with h4 and h5 oligonucleotides. Lane i contains in vitro-transcribed hTR E and N and chimeric hTR-U64 RNA standards. (B) TRAP activity assay of extracts prepared from transiently transfected 293 cells. Oligonucleotides amplifying WT (C3TA2) or AT (A2C4, C2A2C2) telomerase extension products were used as indicated. Each pair of lanes corresponds to 5- and 25-fold dilutions of whole-cell extracts.
- Fig. 5.
hTR expression by Pol III. (A) Schematic of hTR Pol III expression system indicates relative positions of restriction endonuclease sites template residues, H and ACA box residues, sequence tag insertion sites (BalI and StuI), and T→A mutations. (B) Northern blot analysis of total RNA prepared from transiently transfected 293 cells probed with the random hexamer-labeled SE restriction fragment (SE hex). Lane 1, mock transfection; lane 2, empty pRc/CMV vector; lane 3, SE sequence tagged at BalI; lane 4, untagged SE (T41A, T101A); lane 5, untagged SSm (T41A, T101A); lane 6, XE sequence tagged at StuI. Unprocessed (○) and 3′ end-processed (○○) hTR species present in lane 6 are indicated. Lane i contains in vitro-transcribed hTR standards E and N. Non-hTR cross-hybridizing RNAs are indicated by an asterisk. Relative transfection efficiency and RNA recovery were monitored as described in the legend to Fig. 2 (data not shown).
- Fig. 6.
Localization of hTR by subcellular fractionation. (A) Northern blot analysis of RNA prepared from subcellular fractions of HeLa cells. The blot was probed with h4 and h5 oligonucleotides to detect endogenous hTR and the 410-nt RC RNA added during sample preparation to verify loading equivalents and then with oligonucleotides complementary to U3 snoRNA, U2 snRNA, and SRP RNA. Brackets above lanes indicate cell equivalent loadings among the Total, Cytoplasm, and Nuclei lanes and among the Sonicated Nuclei, Nucleoplasm, and Nucleoli lanes. 4× Nucleoli represents 4 cell equivalents of nucleolar RNA relative to the Sonicated Nuclear group. (B) Northern blot analysis of RNA prepared from sucrose gradient fractions following sedimentation of crude HeLa nucleoli, probed as described for panel A. A fraction of total nucleolar RNA is present in lane T.
