Stable expression in yeast of the mature form of human telomerase RNA depends on its association with the box H/ACA small nucleolar RNP proteins Cbf5p, Nhp2p and Nop10p

Abstract

Telomerase is a ribonucleoprotein (RNP) particle required for the replication of telomeres. The RNA component, termed hTR, of human telomerase contains a domain structurally and functionally related to box H/ACA small nucleolar RNAs (snoRNAs). Furthermore, hTR is known to be associated with two core components of H/ACA snoRNPs, hGar1p and Dyskerin (the human counterpart of yeast Cbf5p). To assess the functional importance of the association of hTR with H/ACA snoRNP core proteins, we have attempted to express hTR in a genetically tractable system, Saccharomyces cerevisiae. Both mature non-polyadenylated and polyadenylated forms of hTR accumulate in yeast. The former is associated with all yeast H/ACA snoRNP core proteins, unlike TLC1 RNA, the endogenous RNA component of yeast telomerase. We show that the presence of the H/ACA snoRNP proteins Cbf5p, Nhp2p and Nop10p, but not Gar1p, is required for the accumulation of mature non-polyadenylated hTR in yeast, while accumulation of TLC1 RNA is not affected by the absence of any of these proteins. Our results demonstrate that yeast telomerase is unrelated to H/ACA snoRNPs. In addition, they show that the accumulation in yeast of the mature RNA component of human telomerase depends on its association with three of the four core H/ACA snoRNP proteins. It is likely that this is the case in human cells as well.

Figure 1

Both mature polyA^– hTR RNA and polyA⁺ forms of this RNA can accumulate in S.cerevisiae. Total RNAs (Tot) were extracted from yeast strains transformed either with pFL45esno expression vector lacking insert (lane 1) or with the phTR plasmid, derived from pFL45esno, that carries the DNA fragment encoding hTR (lane 2). A sample of the total RNA preparation obtained from the strain containing phTR was incubated with biotinylated oligo(dT) and polyadenylated RNAs bound to oligo(dT) were precipitated using streptavidin-coated magnetic beads. RNAs were recovered from the supernatant (polyA^–, lane 3) or the pellet (polyA⁺, lane 4). All RNAs were separated on a 6% polyacrylamide gel, transferred to a nylon membrane and hybridized with antisense oligonucleotide probes detecting the TLC1, hTR, snR37 or U3 RNA. Positions of molecular weight markers are indicated on the right.

Figure 2

Mature polyA^– hTR expressed in yeast interacts with all four core proteins of H/ACA snoRNPs. Immunoprecipitation experiments were carried out using IgG–Sepharose and extracts from strains expressing hTR and either Cbf5pZZ (lanes 1 and 2), Gar1pZZ (lanes 3 and 4), Nhp2pZZ (lanes 5 and 6), Nop10pZZ (lanes 7 and 8) or ZZNop1p (lanes 9 and 10). RNAs extracted from one-tenth of the input extracts (I) or from the pellets following immunoprecipitation (P) were separated on a 6% polyacrylamide gel and transferred to a nylon membrane. RNAs indicated on the left were detected by hybridization with antisense oligonucleotide probes.

Figure 3

Accumulation in S.cerevisiae of mature polyA^– hTR requires the presence of Cbf5p, Nhp2p and Nop10p. GAL::cbf5 (lanes 1–5), GAL::gar1 (lanes 6–10), GAL::nhp2 (lanes 11–15) and GAL::nop10 (lanes 16–20) strains transformed with phTR were grown in galactose-containing medium (lanes 1, 6, 11 and 16) and were then transferred to glucose-containing medium for 12, 24, 48 or 72 h. At each time-point, culture samples were collected from which total RNAs were extracted. These were separated on 6% polyacrylamide gels and transferred to nylon membranes. RNAs indicated on the left were detected by hybridization with specific oligonucleotide probes.

Figure 4

Accumulation in S.cerevisiae of polyadenylated forms of hTR and of polyA^– mature TLC1 does not require the presence of Nhp2p. The GAL::nhp2 strain transformed with phTR was grown in galactose-containing medium (lanes 1–3) and was then transferred to glucose-containing medium for 12, 24, 48 and 72 h. Culture samples were collected from which total RNAs were extracted (Tot). Samples of the total RNA preparations were incubated with biotinylated oligo(dT) and polyadenylated RNAs were precipitated using streptavidin-coated magnetic beads. RNAs were recovered from the supernatants (polyA^–) or the pellets (polyA⁺). All RNAs were separated on a 6% polyacrylamide gel, transferred to a nylon membrane and hybridized with antisense oligonucleotide probes detecting TLC1, hTR, snR37 or U3 RNA.