Inverse-folding design of yeast telomerase RNA increases activity in vitro

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 RNAs 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 biological 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 {triangleup}G in 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 and found that it complements a tlc1{triangleup} 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 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.