A functional telomerase RNA swap in vivo reveals the importance of nontemplate RNA domains

Abstract

The ribonucleoprotein (RNP) enzyme telomerase is required for replication of eukaryotic chromosomal termini. The RNA moiety of telomerase is essential for enzyme function and provides the template for telomeric DNA synthesis. However, the roles of its nontemplate domains have not been explored. Here we demonstrate that a novel interspecies telomerase RNA swap in vivo creates a functional but aberrant telomerase. Telomerase RNA from the ciliate Glaucoma chattoni was expressed in Tetrahymena thermophila cells. The telomerase RNAs from these two species have almost superimposable secondary structures. The template region base sequence is identical in the two RNAs, but elsewhere their sequences differ by 49%. This hybrid telomerase RNP was enzymatically active but added only short stretches of telomeric repeat tracts in vivo and in vitro. This new enzyme also had a strong, aberrant DNA cleavage activity in vitro. Thus, molecular interactions in the RNP involving nontemplate RNA domains affect specific aspects of telomerase enzyme function, raising the possibility that they may regulate telomerase activity.

Figure 1

Conserved similarities between T. thermophila and G. chattoni telomerase RNAs (TER1). (A) Schematic summary of the conserved secondary structure between Tetrahymena and Glaucoma TER1 RNAs. Residues in bold represent bases conserved between these two RNAs. Shaded region contains 23 bases of absolute sequence identity centered upon the template region. The templating region contains an RNA sequence specifying the complementary telomeric DNA sequence polymerized at chromosome termini. The position of the C-to-A template base change (43A) is indicated. Despite an overall 35% difference in RNA sequence (49% excluding the template region), both TER1 RNAs fold into similar conformations. (B) Schematic strategy for the heterologous expression of the G. chattoni telomerase RNA gene (Gc.TER1) in T. thermophila cells. A PCR approach was devised to precisely surround the Glaucoma TER1 coding region with Tetrahymena TER1 expression signals producing a chimeric Gc.TER1 gene, which was introduced into wild-type T. thermphila cells.

Figure 2

Heterologous expression and assembly of Glaucoma telomerase RNA into an RNP complex. (A) Northern blot analysis of Tetrahymena transformants expressing the Gc.TER1 gene in vivo. Total RNA from Tetrahymena or Glaucoma TER1 or ter1–43A transformants was fractionated on denaturing gels. Blots probed with a Gc.TER1-specific gene probe (lanes 1–4) revealed presence of Gc.TER1 RNA in vivo (lanes 3 and 4). Blots were reprobed with a Tt.TER1-specific probe (lanes 5–8); endogenous Tetrahymena TER1 RNA was observed in Gc.TER1 and Gc.ter1–43A transformants (lanes 7 and 8). (B) Assembly of Tetrahymena TER1 RNA into a telomerase RNP complex. RNP complexes prepared from untransformed, vegetative Tetrahymena cells (lane 2) and Tt.TER1 or Tt.ter1–43A transformants (lanes 3 and 4) were studied by RNP gel analysis; blots were probed with a Tetrahymena TER1-specific probe (see Methods). Lane 1 contains total RNA (≈10 μg) prepared from Tt.TER1 transformants. Light shaded arrow indicates free telomerase RNA, and dark arrow indicates lower putative telomerase RNP complex. (C) Identity of RNP complexes in Gc.ter1–43A transformants. RNP complexes from Gc.ter1–43A transformants were analyzed by sequentially probing with a Gc.TER1-specific probe (lane 1) and then a Tt.TER1-specific probe (lane 2). Lanes 3 and 4 are duplicate loadings subjected to Western blot analysis using telomerase anti-p80 and -p95 antibodies, respectively. Arrow identifies lower RNP complex.

Figure 3

Southern blot analysis of genomic DNA from T. thermophila transformants containing Tetrahymena and Glaucoma TER1 and ter1–43A genes. PstI-cut transformant genomic DNAs were separated by 0.7% agarose gel electrophoresis and subjected to Southern blot analysis. Transformant macronuclear rDNA telomeres were analyzed using either a G₄T₃- (lanes 1–4) or a G₄T₂-specific (lanes 5–8) oligonucleotide probe (17). Lanes 5–8 are duplicate lanes to lanes 1–4. Only part of the Southern blot containing the rDNA telomeres is shown.

Figure 4

Incorporation of G₄T₃ variant telomeric repeats in vivo and in vitro. (A) DNA sequences of representative G₄T₃-containing telomere clones isolated from Gc.ter1–43A and Tt.ter1–43A transformant telomere libraries. Boxes highlight occurrence of variant G₄T₃ repeats. (B) In vitro telomerase activity from Tt.TER1 (lane 1), Tt.ter1–43A (lane 2), and Gc.ter1–43A (lane 3) extracts in presence of labeled dGTP and unlabeled dTTP. Arrows indicate positions of G₄T₃ repeat products in Glaucoma ter1–43A extract amid the largely wild-type repeat pattern, consistent with only two rounds of 43A-templated elongation synthesis. Size of unlabeled input primer is indicated. (C) In vitro telomerase activity from transformant extracts in the presence of [α-³²P] dTTP-only. Lanes 1, 3, 5, and 7: telomerase fractions were pretreated with RNase A. Lane 2, extension products from Tt.TER1; lane 4, Tt.ter1–43A extracts. Lengths of control DNA fragments are indicated. Schematic summaries represent products predicted from wild-type or 43A telomerase RNA template. The lower band in the n + 1 doublet (arrowed, lanes 6 and 8) likely represents cleavage of the 3′-terminal dG from primer d(T₂G₄)₃ and addition of two radiolabeled dT* bases, producing a d(T₂G₄)₂TTGGGT*T* product. This species is expected to migrate slightly faster than the d(T₂G₄)₂TTGGGGT* product, generated by a single dT* residue addition onto the uncleaved primer.