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Stepwise protein-mediated RNA folding directs assembly of telomerase ribonucleoprotein

Abstract

Telomerase is an essential cellular ribonucleoprotein (RNP) that solves the end replication problem and maintains chromosome stability by adding telomeric DNA to the termini of linear chromosomes1,2,3. Genetic mutations that abrogate the normal assembly of telomerase RNP cause human disease4. It is therefore of fundamental and medical importance to decipher cellular strategies for telomerase biogenesis, which will require new insights into how specific interactions occur in a precise order along the RNP assembly pathway. Here we use a single-molecule approach to dissect the individual assembly steps of telomerase. Direct observation of complex formation in real time revealed two sequential steps of protein-induced RNA folding, establishing a hierarchical RNP assembly mechanism: interaction with the telomerase holoenzyme protein p65 induces structural rearrangement of telomerase RNA, which in turn directs the binding of the telomerase reverse transcriptase to form the functional ternary complex. This hierarchical assembly process is facilitated by an evolutionarily conserved structural motif within the RNA. These results identify the RNA folding pathway during telomerase biogenesis and define the mechanism of action for an essential telomerase holoenzyme protein.

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Figure 1: Telomerase proteins p65 and TERT induce distinct conformational changes in telomerase RNA.
Figure 2: Real-time assembly of individual telomerase RNP complexes.
Figure 3: The p65-induced RNA structural change occurs within stem IV and requires the conserved GA bulge.
Figure 4: The p65 protein and conserved GA bulge in stem IV of telomerase RNA are required for telomerase assembly.

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Acknowledgements

We thank M. Bates for LabView software for data acquisition. This work was supported in part by the NIH and the Packard Foundation (X.Z.), and the NIH (K.C.). X.Z. is a Howard Hughes Medical Institute investigator. M.D.S. is a. NIH Ruth L. Kirschstein NSRA Fellow.

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Correspondence to Xiaowei Zhuang.

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Supplementary information

Supplementary Information

This file contains Supplementary Methods; Supplementary Table 1; Supplementary Figures 1-12 with Legends and additional references. The Supplementary Methods section includes additional details for RNA construct preparation, FRET data acquisition and analysis, telomerase RNP reconstitution and activity assays, and in vivo telomerase RNA accumulation assays. Supplementary Table 1 provides oligonucleotide sequences (not provided in the main text) used in RNA ligation reactions. Supplementary Figures 1-12 describe: RNA construct preparation methodology; FRET data analysis; the binding affinities of p65 and TERT to the telomerase RNA; effects of RNA and protein sequence modification, as well as surface immobilization, on protein binding; telomerase function and FRET measurements; photo-physical controls which rule out possible FRET artifacts derived from protein-dye interaction; FRET measurements on catalytically active telomerase RNP reconstituted in RRL; details of the observed minor fraction RNP assembly behavior; native gel assay for p65-RNA binding, conformational dynamics of telomerase RNA in the presence of TERT alone; and in vivo telomerase RNA accumulationl experiments. (PDF 2789 kb)

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Stone, M., Mihalusova, M., O’Connor, C. et al. Stepwise protein-mediated RNA folding directs assembly of telomerase ribonucleoprotein. Nature 446, 458–461 (2007). https://doi.org/10.1038/nature05600

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