Abstract
Telomerase extends chromosome ends by copying a short template sequence within its intrinsic RNA component. Telomerase RNA (TR) from different groups of species varies dramatically in sequence and size. We report here the bioinformatic identification, secondary structure comparison, and functional analysis of the smallest known vertebrate TRs from five teleost fishes. The teleost TRs (312-348 nucleotides) are significantly smaller than the cartilaginous fish TRs (478-559 nucleotides) and tetrapod TRs. This remarkable length reduction of teleost fish TRs correlates positively with the genome size, reflecting an unusual structural plasticity of TR during evolution. The teleost TR consists of a compact three-domain structure, lacking most of the sequences in regions that are variable in other vertebrate TR structures. The medaka and fugu TRs, when assembled with their telomerase reverse transcriptase (TERT) protein counterparts, reconstituted active and processive telomerase enzymes. Titration analysis of individual RNA domains suggests that the efficient assembly of the telomerase complex is influenced more by the telomerase reverse transcriptase (TERT) binding of the CR4-CR5 domain than the pseudoknot domain of TR. The remarkably small teleost fish TR further expands our understanding about the evolutionary divergence of vertebrate TR.
Highlights
Telomeres are specialized DNA-protein complexes that cap chromosome ends and are important for genome stability and cellular proliferation [1]
We report here the bioinformatic identification, secondary structure comparison, and functional analysis of the smallest known vertebrate Telomerase RNA (TR) from five teleost fishes
The length of telomeric DNA in most eukaryotes is maintained by telomerase, a specialized reverse transcriptase that synthesizes telomeric DNA repeats at chromosome ends to counterbalance the natural shortening that occurs during DNA replication
Summary
Bioinformatics Search of Teleost Fish TR Sequences—A sequence search was performed using fragrep. Zebrafish, and fugu, the PCR products of TR genes were cloned into the EcoRV site of the pZero vector (Invitrogen) to generate pMedaka-TR, pZebrafish-TR, and pFugu-TR. Identification and Cloning of Teleost Fish TERT Genes—To reconstitute telomerase activity, we cloned TERT genes from medaka, zebrafish, and fugu. To clone the TERT genes, the coding sequences of medaka and zebrafish TERT genes were PCR amplified from the cDNA samples prepared from total RNA samples using Thermoscript reverse transcriptase (Invitrogen) and an oligo(dT) reverse primer. The PCR products of the medaka, zebrafish, and fugu TERT cDNAs were cloned into the pCITE vector for in vitro synthesis of the recombinant TERT proteins. Riboprobes with sequences complementary to the target RNA were generated by in vitro transcription from a PCR DNA template that contained the T7 promoter and labeled internally with [␣-32P]UTP using a MaxiScript kit (Ambion). The relative activities were plotted against concentrations of RNA fragment and the nonlinear regression curve fitting was carried out using the one-site binding (hyperbola) equation, Y ϭ Bmaxϫ X/(Kd ϩ X) (Prism 5, Graphpad Software, Inc.)
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