Human telomerase RNA elements

Details Human telomerase RNA elements Human telomerase RNA elements

2000-08-24

2002-01-29

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S325000, C435S375000, C435S007800, C536S023100, C536S023500, C536S024100, C530S350000

Reexamination Certificate

active

06342358

ABSTRACT:

INTRODUCTION
1. Field of the Invention
The field of the invention is functional elements of human telomerase—an enzyme important in cell growth.
2. Background of the Invention
Telomeres are the dynamic nucleoprotein complexes that cap the ends of linear chromosomes. They prevent undesirable chromosome rearrangements and protect against genomic instability and the associated risk of carcinogenesis (Artandi and DePinho, 2000). Telomeres can also function as a mitotic clock that counts cell divisions by the gradual erosion of telomeric sequence. Telomere shortening forces cultured human primary cells to stop dividing when a critical minimum telomere length is reached (Colgin and Reddel, 1999). This entry into senescence acts as a protective checkpoint, guarding against genomic instability induced by telomere loss.
Many cellular factors are required to maintain telomere stability, including the telomere binding proteins that orchestrate a higher order telomeric chromatin structure (Collins, 2000). At a regulatory level, however, changes in telomere length appear to be accomplished primarily by activation or inhibition of telomerase. The telomerase ribonucleoprotein (RNP) extends chromosome 3′ ends by addition of one strand of tandem DNA repeats (Greider, 1995). Telomerases in all species share at least two components essential for catalytic activity: the telomerase reverse transcriptase protein (TERT) and the telomerase RNA (Bryan and Cech, 1999). Although TERTs share the reverse transcriptase (RT) active site motifs of viral RTs (Lingner et al., 1997), they are unique in their stable association with a telomerase RNA that contains the template for telomeric repeat synthesis Greider and Blackburn, 1989). Telomerase RNAs range in size from about 150-200 ucleotides (nt) in ciliates to greater than 1000 nt in yeasts.
In humans, misregulation of telomerase activity can have dire consequences. Telomerase activation accompanies tumorigenesis and is important for the continued viability of cultured human tumor cells (Kim et al., 1994; Artandi and DePinho, 2000; Collins, 2000). However, recent studies have suggested that telomerase activation in at least some human cell types is also essential for normal growth and development. Premature mortality caused by the X-linked disease dyskeratosis congenita (DKC) results from proliferative deficiencies and an increased risk of cancer in tissues which are normally highly regenerative, such as the skin and blood (Dokal, 1999). Cells from DKC patients have reduced levels of telomerase RNP and hastened telomere shortening (Mitchell et al., 1999b). This suggests that telomerase activation in highly proliferative tissues may be necessary to suppress potential genomic instability and to guarantee enough renewal capacity for a typical human lifespan. Together, these findings reveal that telomerase activation and inhibition must be carefully balanced to meet the proliferative demands of normal cells while at the same time guarding against the potential for unbridled proliferation of tumors. As a result, pharmacological methods for activating or restraining telomerase activity in vivo would both be useful.
The human telomerase RNA (hTR) is transcribed by RNA polymerase II and processed at its 3′ end to yield a mature transcript of 451 nt (Feng et al., 1995; Zaug et al., 1996; Mitchell et al., 1999a). The template for reverse transcription lies near the 5′ end of the molecule and specifies incorporation of the sequence TTAGGG to chromosome ends. In a previous study, we identified hTR primary sequence elements that are required for the stability and 3′ end processing of recombinant hTR in vivo (Mitchell et al., 1999a). Surprisingly, these elements form part of a structural motif shared with H/ACA small nucleolar (sno)RNAs, an RNA family that functions in the maturation of ribosomal RNA by directing cleavage and pseudouridine (&PSgr;) formation (Tollervey and Kiss, 1997). Hybridization of a snoRNA to target RNA specifies the site of modification, while protein components of the stable snoRNP catalyze the reaction itself. Phylogenetic comparison of 35 vertebrate telomerase RNAs confirmed that the H/ACA motif is a universally conserved feature that is not present in ciliate or yeast telomerase RNAs (Chen et al., 2000). The 3′ half of hTR as bounded by the elements of the H/ACA motif (nt 211-451) can accumulate independently of the full-length molecule in vivo (Mitchell et al., 1999a). We refer to this region as the hTR H/ACA domain (see FIG.
1
). When the hTR H/ACA domain is replaced with a heterologous H/ACA snoRNA, the chimeric RNA accumulates but does not support telomerase activity (Mitchell et al., 1999a). Therefore, sequences within the hTR H/ACA domain are critical for telomerase activity independent of the requirement for hTR stability in vivo.
Here, we define distinct motifs within the human telomerase RNA that contribute to telomerase RNP accumulation and activity. We find that hTR precursor processing, mature RNA accumulation, and H/ACA protein association are inseparably linked and require the consensus H/ACA motif elements within the H/ACA domain. Furthermore, we demonstrate that two regions within the telomerase RNA are required for telomerase activity in vivo and in vitro. One of these regions contains the template for reverse transcription as expected (hTR nt 1-209); the other is a telomerase-specific element within the H/ACA domain (hTR nt 241-330). Surprisingly, we find that both of these regions interact independently with TERT and bind to TERT in a largely noncooperative manner. Thus, a vertebrate-specific telomerase RNA motif physically separable from the template is required for telomerase activity. This work reveals an unexpected functional requirement for two distinct telomerase RNA-TERT interactions within the same telomerase RNP and establishes a fundamental difference between the structure of ciliate and vertebrate telomerase RNPs.
Relevant Literature
See U.S. Pat. Nos. 5,917,025 and 5,770,422.
SUMMARY OF THE INVENTION
Telomerase inhibition has utility as a clinical treatment for a broad range of human cancers (treated by telomerase inhibition) and age- or disease-induced cellular proliferative deficiencies (treated by telomerase activation). Requirements for telomerase function at a structural level have hitherto remained largely unknown. Here, we demonstrate the structural requirements for function of the essential human telomerase RNA component (hTR) in vivo and in vitro. Two types of function for RNA elements are discriminated. First, we have identified RNA elements that are essential for RNA stability in vivo but are dispensable for catalytic activity in vitro. Second, we have identified RNA motifs that are critical for catalytic activity in vivo and in vitro.
The first category, RNA elements essential for RNA stability in vivo, includes all elements of the consensus H/ACA motif in proper sequence context (5′ terminal stem: nts 211-214 paired to 367-370; H box: unpaired nts 372-377; 3′ terminal stem: nts 381-384 paired to 440-443; ACA box: unpaired nts 446-448) and one additional element (3′ stem-loop: nts 411-418). Cellular accumulation of stable hTR, dependent on the H/ACA motif elements described above, is coincident with the association of RNA with H/ACA proteins (as assayed by association with the protein dyskerin, as a cooperative assembly of the proteins dyskerin, hNhp2, hNop10 and additional cooperative or noncooperative assembly of the protein hGAR1.
The second category, RNA elements essential for catalytic activity both in vivo and in vitro includes a region containing the template (nts 1-208 in vivo and in vitro; in vitro element minimized to nts 44-186) and a second region termed IH1 (nts 241-330 in vivo and in vitro; in vitro element minimized to 253-322 without 271-285). Each of these RNA elements binds to the telomerase reverse transcriptase protein (TERT) independently, both in vivo and in vitro. However, binding of both elements is required for catalytic activity

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