Engineering cis-telomerase RNAs that add telomeric repeats to themselves

Abstract

Telomerase is a ribonucleoprotein complex consisting of a protein reverse transcriptase (TERT) and an RNA subunit (TR). Telomerase normally adds telomeric DNA repeats to chromosome ends. Here, we engineer human and Tetrahymena cis-telomerase RNAs, each having a DNA primer covalently linked to its 3′ end. We find that cis-telomerase synthesizes DNA with increased repeat addition processivity (RAP) but does not completely rescue the RAP defect of the L14A mutant of Tetrahymena TERT. This supports the conclusion that L14 has a function beyond binding the DNA primer and preventing dissociation during multiple rounds of repeat addition. By comparing cis-telomerases with various linker lengths, we find that a 5 nt linker gives near-optimal activity, indicating that the distance between the 3′ end of the telomerase RNA pseudoknot region and the 5′ end of the DNA primer is ∼33 Å. Even a 2 nt linker (∼14 Å) gives some activity, indicating a high degree of conformational flexibility in this ribonucleoprotein complex. More generally, the cis system will allow structure-function relationships of each RNA molecule to be read directly through the reaction that it performs on itself.ribonucleoprotein complex | processivity | reverse transcriptase | telomerase N atural RNAs involved in biocatalysis act either "in cis" (intramolecular reactions) or "in trans" (intermolecular reactions). Cis-acting RNAs are exemplified by the self-splicing group I and group II introns and the self-cleaving hammerhead and hairpin ribozymes (1, 2). Trans-acting RNAs include: the ribosomal RNAs that use aminoacylated tRNAs as substrates (3), the spliceosomal small nuclear RNAs that use pre-mRNAs as substrates (4), and the RNase P ribozyme that uses tRNA precursors containing 5′-leader sequences as substrates (5). In a number of cases, cis-acting ribozymes have been converted into trans-acting RNA enzymes that splice or cleave exogenous RNA substrates. The incentive for engineering these trans-acting ribozymes was initially to demonstrate that they were true catalysts, capable of multiple-turnover reactions. A subsequent rationale was to create RNA-cleaving agents for biotechnology. Transformation in the other direction, from trans-acting to cis-acting RNAs, has been accomplished for RNase P. By engineering a chimeric RNA that joins the RNase P RNA to its pre-tRNA substrate, pre-tRNA-RNase P RNA conjugates were designed to undergo accurate and efficient self-cleavage (6, 7). In the present work, we describe the conversion of the telomerase RNA-TERT complex, which normally acts in trans to extend telomeric DNA primers, into a cis-acting system that extends its own 3′ end. Thus, a trans-acting RNA can be converted into a cis-acting RNA even in the context of a complex ribonucleoprotein (RNP).The telomerase RNP uses its reverse transcriptase subunit (TERT) to copy a template sequence within its intrinsic RNA subunit (TR), thereby adding telomeric DNA repeats to the ends of chromosomes (8, 9). Although TERT has clear sequence conservation among divergent...

Structural basis for protein-RNA recognition in telomerase

“…5b). In addition, Qiao et al 32 previously reported that an engineered human cis -telomerase that linked the 3′ end of the telomerase PK region to the 5′ end of the DNA primer with a five-nucleotide linker could generate a near-optimal telomerase activity, thus indicating that the distance between the 3′ end of PK and the 5′ end of DNA primer is ~33 Å. In keeping with this observation, the orientation of PK in our proposed model could allow the 3′ end of PK to be adjacent to the 5′ end of the DNA (Supplementary Fig.…”

Isolation and biological characterization of chicken amnion epithelial cells

“…13 Cancer cells and ESCs express this marker, which indicates the potential for telomerase activators to contribute to the development of tumors. 14 Our finding that AECs did not express TERT suggests that AECs do not form tumors.…”

“…TERT is an enzyme that adds DNA sequence repeats (“TTAGGG” in all vertebrates) to the 3' end of DNA strands in the telomere regions, which are found at the ends of eukaryotic chromosomes. 13 Cancer cells and ESCs express this marker, which indicates the potential for telomerase activators to contribute to the development of tumors. 14 Our finding that AECs did not express TERT suggests that AECs do not form tumors.…”

Structurally conserved five nucleotide bulge determines the overall topology of the core domain of human telomerase RNA

“…This region of the pseudoknot is more protected from chemical probing in vivo than the free TR is in vitro (46). An engineered cis-telomerase, in which a 6 nt DNA primer is linked to the 3′ end of the pseudoknot, has its maximal activity with a linker length of 5 nt or longer, which corresponds to a maximal distance of 33 Å between the end of the pseudoknot and the 5′ end of the TR template (47). This result is consistent with the close proximity of the end of the pseudoknot to the template seen in our model structure.…”