Lebo Kevin J, Zappulla David C
Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015, USA.
Noncoding RNA. 2023 Aug 28;9(5):51. doi: 10.3390/ncrna9050051.
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 feature of 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 ∆ 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 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 that could be useful for controlling RNA structure in basic research and biomedicine.
端粒酶RNA，即TLC1，是一种1157个核苷酸的非编码RNA，它既是DNA合成的模板，也是端粒酶RNP全酶蛋白亚基的柔性支架。易于操作的芽殖酵母系统为体内端粒生物学提供了具有里程碑意义的发现，但酵母端粒酶研究受到了以下事实的阻碍：大型TLC1 RNA亚基在体外不能支持强大的端粒酶活性。相比之下，包含催化核心结构域且缺少RNA长臂的155 - 500个核苷酸的小型化TLC1等位基因确实能重建强大的活性。我们推测全长TLC1在体外容易错误折叠。为了创建一个预测能折叠成其生物学相关结构的全长酵母端粒酶RNA，我们采用了反向RNA折叠方法，根据软件特征改变了59个核苷酸，这些核苷酸预计会增加折叠成模拟天然结构的能量偏好性。与野生型相比，序列变化使这个“确定臂”等位基因DA - TLC1的预测∆G降低了61千卡/摩尔（-19%）。我们测试了DA - TLC1的重建活性，发现它比野生型TLC1的活性强约5倍，这表明反向折叠设计确实改善了体外折叠成催化活性构象的情况。我们还测试了DA - TLC1在体内是否起作用，发现它能补充∆菌株，使细胞避免衰老并维持接近野生型长度的端粒。然而，我们测试的所有反向设计的RNA在体内的丰度都降低了。特别是，几乎对所有Ku臂进行反向设计都会导致细胞中端粒酶RNA丰度大幅降低以及端粒非常短。总体而言，这些结果表明端粒酶RNA的反向设计增加了体外活性，同时降低了体内丰度。这项研究提供了一种经过生物化学和生物学测试的利用反向设计RNA的方法，这可能有助于在基础研究和生物医学中控制RNA结构。
