Aug 2, 1996 - telomerase, the addition of in vitro transcribed hTR reconstituted telomerase activity. The activity was dependent upon and specific to hTR.
The EMBO Journal vol.15 no.21 pp.5928-5935, 1996
Reconstitution of human telomerase activity and identification of a minimal functional region of the human telomerase RNA Chantal Autexier1, Ronald Pruzan1 2, Walter D.Funk2 and Carol W.Greider1l3 'Cold Spring Harbor Laboratory, PO Box 100, Cold Spring Harbor, NY 11724 and 2Geron Corporation, 200 Constitution Drive, Menlo Park, CA 94025, USA 3Corresponding author C.Autexier and R.Pruzan contributed equally to this work
Telomerase is a ribonucleoprotein that catalyzes telomere elongation through the addition of TTAGGG repeats in humans. Activation of telomerase is often associated with immortalization of human cells and cancer. To dissect the human telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential 445 nucleotide human telomerase RNA (hTR) by micrococcal nuclease digestion of partially purified human telomerase, the addition of in vitro transcribed hTR reconstituted telomerase activity. The activity was dependent upon and specific to hTR. Using this assay, truncations at the 5' and 3' ends of hTR identified a functional region of hTR, similar in size to the fulllength telomerase RNAs from ciliates. This region is located between positions 1-203. Furthermore, we found that residues 144, 5' to the template region (residues 46-56) are not essential for activity, indicating a minimal functional region is located between residues 44-203. Mutagenesis of full-length hTR between residues 170-179, 180-189 or 190-199 almost completely abolished the ability of the hTR to function in the reconstitution of telomerase activity, suggesting that sequences or structures within this 30 nucleotide region are required for activity, perhaps by binding telomerase protein components. Keywords: human/micrococcal nuclease/reconstitution/
ribonucleoprotein/telomeres
Introduction Telomerase is an enzyme essential for telomere length maintenance (Yu et al., 1990; Singer and Gottschling, 1994; McEachern and Blackburn, 1995). Conventional DNA polymerases cannot complete the replication of chromosome ends, and without a mechanism to overcome the end replication problem, chromosomes are predicted to shorten with each round of cell division (Watson, 1972; Olovnikov, 1973). Telomerase is a specialized telomerespecific polymerase that counteracts this end shortening through de novo sequence addition, using a sequence in its RNA component as a template. Telomerase activity was first identified in the unicellular ciliated protozoa Tetrahymena (Greider and Blackburn, 1985). The genes
5928
encoding the RNA and protein components of telomerase were also first cloned from
Tetrahymena (Greider and
Blackburn, 1989; Collins et al., 1995). Activity and/or RNA components have since been identified in other
ciliates, in human, mouse, Xenopus and yeast (for review see Greider, 1996). The protein components from these other organisms have not yet been identified. In immortal single cell eukaryotes a steady state equilibrium of telomere length is established which is regulated by a number of genes in addition to telomerase (Greider, 1996). Telomere length is not maintained in primary human somatic cells. Primary human cells have a limited replicative lifespan in culture and telomere shortening correlates with loss of replicative capacity (Harley et al., 1990; Lindsey et al., 1991; Allsopp et al., 1992; Vaziri et al., 1993). Telomere lengths in Wilms' tumors, mammary and colon carcinomas are shorter than in the normal
neighboring tissues (de Lange et al., 1990; Hastie et al., 1990), and short telomeres have subsequently been seen in a number of different tumor types (reviewed in Bacchetti and Counter, 1995; Wright and Shay, 1995; Bacchetti, 1996; Greider and Harley, 1996). Using a sensitive PCR-based telomerase activity assay, telomerase activity was not detected in a large number of primary cell strains and human tissues (Kim et al., 1994). In contrast to many normal human cells, tumor-derived cell lines and ~90% of primary tumor samples contain detectable telomerase activity, indicating that telomerase activation in tumors may be very common (Kim et al., 1994; for recent reviews see Wright and Shay, 1995; Bacchetti, 1996; Greider and Harley, 1996). The presence of telomerase activity and short telomeres in tumors suggests that inhibiting telomerase may be an effective cancer treatment (Harley et al., 1994). To understand the role of telomerase in human cancer, it is important to understand its role in telomere length regulation, how it is regulated and its mechanism of action. To date, only the RNA component of human telomerase has been identified (Feng et al., 1995). To define the functional regions of the human telomerase RNA component (hTR), we developed a reconstitution assay for telomerase activity based on the addition of synthetic hTR to a micrococcal nuclease (MNase)-treated human extract. This assay was modified from the reconstitution assay developed for Tetrahymena telomerase (Autexier and Greider, 1994). The reconstruction of functional ribonucleoproteins such as ribosomes, ribonuclease P, signal recognition particle, small nuclear RNPs and Tetrahymena telomerase has facilitated a detailed understanding of the function, structure and assembly of these enzymes (Lumelsky and Altman, 1988; Nierhaus, 1991; Strub et al., 1991; Autexier and Greider, 1994, 1995; Segault et al., 1995). Reconstitution of Tetrahymena telomerase activity with mutant telomerase RNA has allowed the identification © Oxford University Press
Telomerase reconstitution and hTR deletion analysis
of a flexible alignment region for substrate and/or product recognition (Autexier and Greider, 1994, 1995). This assay has also enabled the dissection of a conserved region upstream of the template domain (Romero and Blackburn, 1991), whose location and sequence, in the Tetrahymena telomerase RNA, defines the 5' boundary of the template region (Autexier and Greider, 1995). Under appropriate conditions, human telomerase activity can be restored with in vitro transcribed hTR. The reconstituted activity was dependent upon and specific to hTR. Using this assay we defined a minimal functional region of hTR that is the size of full-length ciliate telomerase RNAs. The ability to reconstitute telomerase activity from synthetic RNA and partially purified human extracts should facilitate the identification of human telomerase protein components and the functional analysis of human telomerase.
5S RNiA --x. -
v
-
nn c ri e
e2*iA
-n ceextrat
v
6
2_
-1NA
-TR w
1 E.. a
rR IA1 .
E c
s
0.8
0.8
6
12
_
_
.
12
6
.` 2v
Results Reconstitution of human telomerase activity To determine whether human telomerase activity could be reconstituted using a synthetic RNA, partially purified human telomerase extracts were first treated with MNase to remove endogenous telomerase RNA. Telomerase activity was followed by a modification of the telomere repeat amplification protocol (TRAP) assay (Kim et al., 1994) (see Materials and methods). MNase digestion abolished endogenous telomerase activity. When no RNA was added to the MNase-treated extract, telomerase activity was not restored (Figure 1). Northern analysis of the MNasetreated extract indicated that only fragments of hTR
