Conserved N-terminal motifs of telomerase reverse transcriptase required for ribonucleoprotein assembly in vivo.
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.
Bosoy
Dimitry
D
Department of Microbiology and Immunology, W. R. Hearst Microbiology Research Center, Weill Medical College of Cornell University, New York, New York 10021, USA.
Peng
Yun
Y
Mian
I Saira
IS
Lue
Neal F
NF
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004268:DNA-Binding Proteins; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics; D012261:Ribonucleoproteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019098:Telomerase / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D012261:Ribonucleoproteins; D012313:RNA; D019098:Telomerase