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RNA Binding Domain of Telomerase Reverse Transcriptase Cary Lai University of San Francisco, [email protected]

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RNA Binding Domain of Telomerase Reverse Transcriptase Cary Lai University of San Francisco, Cklai2@Usfca.Edu The University of San Francisco USF Scholarship: a digital repository @ Gleeson Library | Geschke Center Biology Faculty Publications Biology 2001 RNA Binding Domain of Telomerase Reverse Transcriptase Cary Lai University of San Francisco, [email protected] James R. Mitchell Kathleen Collins Follow this and additional works at: http://repository.usfca.edu/biol_fac Part of the Biology Commons Recommended Citation Cary K. Lai, James R. Mitchell, and Kathleen Collins. RNA Binding Domain of Telomerase Reverse Transcriptase. Mol Cell Biol. 2001 Feb; 21(4): 990–1000. This Article is brought to you for free and open access by the Biology at USF Scholarship: a digital repository @ Gleeson Library | Geschke Center. It has been accepted for inclusion in Biology Faculty Publications by an authorized administrator of USF Scholarship: a digital repository @ Gleeson Library | Geschke Center. For more information, please contact [email protected]. MOLECULAR AND CELLULAR BIOLOGY, Feb. 2001, p. 990–1000 Vol. 21, No. 4 0270-7306/01/$04.00ϩ0 DOI: 10.1128/MCB.21.4.990–1000.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved. RNA Binding Domain of Telomerase Reverse Transcriptase CARY K. LAI, JAMES R. MITCHELL, AND KATHLEEN COLLINS* Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3204 Received 23 October 2000/Returned for modification 20 November 2000/Accepted 28 November 2000 Telomerase is a ribonucleoprotein reverse transcriptase that extends the ends of chromosomes. The two telomerase subunits essential for catalysis in vitro are the telomerase reverse transcriptase (TERT) and the telomerase RNA. Using truncations and site-specific mutations, we identified sequence elements of TERT and telomerase RNA required for catalytic activity and protein-RNA interaction for Tetrahymena thermophila telo- merase. We found that the TERT amino and carboxyl termini, although evolutionarily poorly conserved, are none- theless important for catalytic activity. In contrast, high-affinity telomerase RNA binding requires only a small region in the amino terminus of TERT. Surprisingly, the TERT region necessary and sufficient for telomerase RNA binding is completely separable from the reverse transcriptase motifs. The minimal Tetrahymena TERT RNA binding domain contains two sequence motifs with ciliate-specific conservation and one TERT motif with conservation across all species. With human TERT, we demonstrate that a similar region within the TERT amino terminus is essential for human telomerase RNA binding as well. Finally, we defined the Tetrahymena telomerase RNA sequences that are essential for TERT interaction. We found that a four-nucleotide region 5؅ of the template is critical for TERT binding and that the 5؅ end of telomerase RNA is sufficient for TERT binding. Our results reveal at least one evolutionarily conserved molecular mechanism by which the telomerase reverse transcriptase is functionally specialized for obligate use of an internal RNA template. Telomeres are the structures at chromosome ends that dis- cific features of activity such as nucleotide and repeat addition tinguish chromosome termini from double-stranded DNA processivities. These results suggest that the RNA plays roles breaks, protecting natural chromosome ends against fusion, in addition to simply providing the template sequence. Ciliate recombination, and degradation (reviewed in reference 35). In telomerase RNAs have limited primary sequence homology most eukaryotes, telomeric DNA is composed of a tandem but do share elements of evolutionarily conserved secondary array of simple sequence repeats. Some of these repeats are structure (20, 22, 23, 29). lost with each round of chromosome duplication, requiring a Only one protein, the telomerase RT (TERT), is known to separate DNA synthesis mechanism to compensate for this be common to telomerase RNPs of all species (reviewed in loss. Most commonly, this task is accomplished by the enzyme reference 4). The central region of the TERT polypeptide telomerase. Telomerase is a reverse transcriptase (RT) that contains RT active site motifs which are essential for activity can add telomeric repeats to chromosome ends de novo by in vivo and in vitro (21, 33). Aside from these RT motifs, only copying a template region within its integral RNA (reviewed in limited patches of TERT-specific sequence similarity have reference 13). Telomerase activity is required for the sustained been identified (6, 24, 28, 34). It is possible to assemble an ac- growth of most single-celled eukaryotes and immortal cell tive recombinant telomerase RNP containing only TERT and lines, including human tumor cells (15, 16, 37). Insufficient telomerase RNA, bypassing the cellular requirement for other telomerase activity induces the replicative senescence of pri- telomerase proteins and a specific telomerase RNP assembly mary human cells in culture (3) and may contribute to age- pathway (reviewed in reference 9). Expression of TERT and dependent decreases in the renewal capacity of certain human telomerase RNA from human, mouse, or the ciliate Tetrahy- tissues (27). mena thermophila in rabbit reticulocyte lysate is sufficient for One of the unique features of telomerase as an RT is that telomerase activity (10, 12, 33). This catalytic core of telome- the assembly of a stable ribonucleoprotein (RNP) complex is rase faithfully positions the template to direct accurate telo- required for polymerase activity. Thus, unlike viral RTs, telo- meric repeat synthesis. Notably, the assembly of recombinant merase recognizes a template containing RNA with sequence human or Tetrahymena telomerase RNPs involves the partici- specificity. The roles of the telomerase RNA component in pation of other activities in reticulocyte lysate (17, 19). Recom- enzyme function have been most thoroughly studied in ciliate binant Tetrahymena telomerase RNP assembly occurs with high telomerases (reviewed in reference 8). Template changes alter specificity and is robust enough to allow detection of telome- the sequence of the synthesized telomeric repeat, as expected. rase RNA coimmunoprecipitated with TERT (19). In addition, sequence substitutions both inside and outside the Tetrahymena template affect RNP assembly, overall activity level, and spe- Here, we have used recombinant telomerase reconstituted in rabbit reticulocyte lysate to investigate the functional significance of regions within TERT and the telo- merase RNA. We find that telomerase RNP activity in vitro * Corresponding author. Mailing address: Department of Molecular requires even the TERT extreme N and C termini, which lack and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA 94720-3204. Phone: (510) 643-1598. Fax: (510) 642-6062. E-mail: any substantial evolutionary sequence conservation. In con- [email protected]. trast, the stable interaction of TERT and telomerase RNA 990 VOL. 21, 2001 TERT RNA BINDING DOMAIN 991 requires a surprisingly small region of the TERT N terminus, 10 ␮g of total yeast RNA, and 15 ␮l of a bead slurry in a 350-␮l final volume and completely separable from the RT motifs. The Tetrahymena mixed for1hatroom temperature or overnight at 4°C. After resin washing in binding buffer, one-third of the sample was mixed with sodium dodecyl sulfate- TERT RNA binding domain contains one motif present in all polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, boiled, and an- TERTs (T motif) and two motifs that are ciliate specific (CP alyzed by electrophoresis on SDS–15% PAGE gels. The remaining two-thirds of and CP2 motifs). We find that a similar region of human the sample was extracted with phenol-chloroform and precipitated with ethanol. TERT (hTERT), preceding the RT motifs and containing the Purified nucleic acid was resolved by denaturing gel electrophoresis ina6or9% ϫ T motif, also mediates human telomerase RNA (hTR) binding. denaturing gel (19:1 acrylamide:bis, 7 M urea, 0.6 Tris-borate-EDTA) and transferred to Hybond Nϩ (Amersham) for hybridization analysis. The RNA For the Tetrahymena telomerase RNA, TERT interaction re- blots shown in Fig. 3 and 4B and C were probed with oligonucleotide 8: GAA quires only residues at the 5Ј end of the RNA, including four GGTTATATCAGCACTAGATTT. The RNA blots shown in Fig. 6 and 7 were nucleotides that are predicted to be unpaired. These results probed with oligonucleotide 11: TGACAGTTCTATTACAGATCTG. The RNA Ј have implications for the evolution of telomerase functional blot shown in Fig. 4D was probed for the 3 tag of the telomerase RNA with the oligonucleotide CTACGCCCTTCTCAGAATTCAATA. The quantification of specialization and for the novel mechanism of template defi- 35S protein on SDS-PAGE gels and the quantification of RNA on RNA blots was nition within the telomerase RNP. performed by phosphorimager (Fuji) analysis. Immunoprecipitation of hTERT-hTR complexes. Calcium phosphate-medi- ated transient transfection of adenovirus-transformed human embryonic kidney MATERIALS AND METHODS (293) cells, freeze-thaw lysis and extract preparation, total RNA preparation by Expression constructs. N-terminal TERT truncation expression constructs acid guanidine thiocyanate-phenol-chloroform extraction, RNA blot hybridiza- were created by PCR of the desired TERT coding region followed by cloning tion, immunoprecipitation on Flag antibody resin (Sigma), and hTERT immu- into pCITE4a (Novagen). C-terminal truncation expression constructs were cre- noblots
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