Conserved N-terminal Motifs of Telomerase Reverse Transcriptase Required for Ribonucleoprotein Assembly in Vivo*
- From the ‡Department of Microbiology and Immunology, W. R. Hearst Microbiology Research Center, Weill Medical College of Cornell University, New York, New York 10021 and§Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720
Abstract
Telomerase is a ribonucleoprotein (RNP) reverse transcriptase responsible for the maintenance of one strand of the telomere terminal repeats. The key protein subunit of the telomerase complex, known as TERT, possesses reverse transcriptase (RT)-like motifs that directly mediate nucleotide addition. The RT motifs are located in the C-terminal region of the polypeptide. Sequence alignments also revealed the existence of four conserved motifs (named GQ, CP, QFP, and T) in the N-terminal region of TERT. The GQ motif of yeast TERT has been demonstrated previously to be essential for telomerase catalysis and may participate in RNP formation. In this report, we show that substitution of conserved residues in the CP, QFP, and T motifs of yeast TERT also impairs both telomere maintenance and telomerase activity, thus confirming the validity of the sequence alignment. The extent of telomere shortening correlates with the extent of reduction in the level of telomerase activity, TERT protein, and TERT-associated TLC1 RNA. Overexpression of the mutant proteins does not result in telomere shortening, implying that assembly rather than catalytic function was affected. This notion was further supported by comparing the efficiency of RNP formation in the wild type and the overexpression strains. Taken together, our results show that three of the four N-terminal motifs are required for efficient telomerase RNP formation in vivo but not for the enzymatic function of telomerase. We also show that the majority of telomerase-associated TLC1 RNA has a more upstream 3′ end than previously reported, consistent with additional processing events during RNP maturation.
Footnotes
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↵* This work was supported by Grant R01 GM62631-02 from the National Institutes of Health (to N. L.) and the California Breast Cancer Research Program (to I. S. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, W. R. Hearst Microbiology Research Center, Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021. Tel.: 212-746-6506; Fax: 212-746-8587; E-mail: nflue@mail. med.cornell.edu.
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Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M210645200
- Abbreviations:
- RNP
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ribonucleoprotein
- RT
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reverse transcriptase
- TERTs
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telomerase reverse transcriptases
- nt
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nucleotide
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- Received October 17, 2002.
- Revision received November 25, 2002.
- The American Society for Biochemistry and Molecular Biology, Inc.
