A critical stem-loop structure in the CR4-CR5 domain of mammalian telomerase RNA.
Telomerase is an enzyme that maintains telomere length by adding telomeric sequence repeats onto chromosome ends. The telomerase ribonucleoprotein complex consists of two essential components, a reverse transcriptase and an RNA molecule that provides the template for telomeric repeat synthesis. A common secondary structure of vertebrate telomerase RNA has been proposed based on a phylogenetic comparative analysis of 35 sequences. Here we report the identification of an additional essential base-paired region in the CR4-CR5 domain of mammalian telomerase RNA, termed P6.1. Mouse telomerase RNAs with mutations that disrupted base pairings in the P6.1 helix were unable to reconstitute telomerase activity in vivo. In contrast, an RNA mutant with compensatory mutations that restored base pairings in the P6.1 helix restored telomerase activity. In an in vitro reconstitution system stable base pairing of the P6.1 stem was required for the RNA-protein interaction between the CR4-CR5 domain and the telomerase reverse transcriptase (TERT) protein. Interestingly, two RNA mutations, one that extends the P6.1 stem and one that alters the conserved nucleotides of the L6.1 loop, allowed RNA-protein binding but significantly impaired telomerase activity. These data establish the presence of the P6.1 stem-loop and its importance for the assembly and enzymatic activity of the mammalian telomerase complex.
Chen
Jiunn-Liang
JL
Department of Molecular Biology and Genetics, 725 North Wolfe Street, Hunterian 617, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Opperman
Kay Keyer
KK
Greider
Carol W
CW
D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins; D005347:Fibroblasts; D008322:Mammals / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D019098:Telomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase; C509187:Tert protein, mouse