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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 chromosomes. Genetic mutations that abrogate the normal assembly of telomerase RNP cause human disease. 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.

SUBMITTER: Stone MD

PROVIDER: S-EPMC2720403 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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Stepwise protein-mediated RNA folding directs assembly of telomerase ribonucleoprotein.

Stone Michael D MD Mihalusova Mariana M O'connor Catherine M CM Prathapam Ramadevi R Collins Kathleen K Zhuang Xiaowei X

Nature 20070225 7134

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 chromosomes. Genetic mutations that abrogate the normal assembly of telomerase RNP cause human disease. 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 R ...[more]

PMID: 17322903

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Project description:Saccharomyces cerevisiae telomerase RNA, TLC1, is an 1157 nt non-coding RNA that functions as both a template for DNA synthesis and a flexible scaffold for telomerase RNP holoenzyme protein subunits. The tractable budding yeast system has provided landmark discoveries about telomere biology in vivo, but yeast telomerase research has been hampered by the fact that the large TLC1 RNA subunit does not support robust telomerase activity in vitro. In contrast, 155-500 nt miniaturized TLC1 alleles comprising the catalytic core domain and lacking the RNA's long arms do reconstitute robust activity. We hypothesized that full-length TLC1 is prone to misfolding in vitro. To create a full-length yeast telomerase RNA, predicted to fold into its biologically relevant structure, we took an inverse RNA-folding approach, changing 59 nucleotides predicted to increase the energetic favorability of folding into the modeled native structure based on the p-num feature of Mfold software. The sequence changes lowered the predicted ∆G of this "determined-arm" allele, DA-TLC1, by 61 kcal/mol (-19%) compared to wild-type. We tested DA-TLC1 for reconstituted activity and found it to be ~5-fold more robust than wild-type TLC1, suggesting that the inverse-folding design indeed improved folding in vitro into a catalytically active conformation. We also tested if DA-TLC1 functions in vivo, discovering that it complements a tlc1∆ strain, allowing cells to avoid senescence and maintain telomeres of nearly wild-type length. However, all inverse-designed RNAs that we tested had reduced abundance in vivo. In particular, inverse-designing nearly all of the Ku arm caused a profound reduction in telomerase RNA abundance in the cell and very short telomeres. Overall, these results show that the inverse design of S. cerevisiae telomerase RNA increases activity in vitro, while reducing abundance in vivo. This study provides a biochemically and biologically tested approach to inverse-design RNAs using Mfold that could be useful for controlling RNA structure in basic research and biomedicine.

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Stepwise protein-mediated RNA folding directs assembly of telomerase ribonucleoprotein.

Publications

Stepwise protein-mediated RNA folding directs assembly of telomerase ribonucleoprotein.

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