酵母端粒酶RNA的反向折叠设计可提高体外活性。

IF 3.6 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Non-Coding RNA Pub Date : 2023-08-28 DOI:10.3390/ncrna9050051
Kevin J Lebo, David C Zappulla
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引用次数: 0

摘要

酿酒酵母端粒酶RNA,TLC1,是一种1157nt的非编码RNA,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。易于处理的出芽酵母系统为体内端粒生物学提供了里程碑式的发现,但酵母端粒酶研究受到了阻碍,因为大的TLC1 RNA亚基在体外不支持强大的端粒酶活性。相反,155-500个包含催化核心结构域且缺乏RNA长臂的小型化TLC1等位基因确实重建了强大的活性。我们假设全长TLC1在体外容易发生错误折叠。为了创建全长酵母端粒酶RNA,预测其折叠成其生物学相关结构,我们采用了反向RNA折叠方法,根据Mfold软件的p-num特征,改变59个预测的核苷酸,以增加折叠成模拟天然结构的能量偏好。与野生型相比,序列变化使该“确定臂”等位基因DA-TLC1的预测∆G降低了61 kcal/mol(-19%)。我们测试了DA-TLC1的重组活性,发现它比野生型TLC1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能力。我们还测试了DA-TLC1是否在体内发挥作用,发现它与TLC1∆菌株互补,使细胞避免衰老,并保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在体内的丰度都有所降低。特别是,对几乎所有Ku臂进行逆向设计,导致细胞中端粒酶RNA丰度大幅降低,端粒极短。总之,这些结果表明,酿酒酵母端粒酶RNA的反向设计在体外增加了活性,而在体内降低了丰度。这项研究提供了一种使用Mfold反向设计RNA的生物化学和生物学测试方法,可用于基础研究和生物医学中控制RNA结构。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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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 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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来源期刊
Non-Coding RNA
Non-Coding RNA Biochemistry, Genetics and Molecular Biology-Genetics
CiteScore
6.70
自引率
4.70%
发文量
74
审稿时长
10 weeks
期刊介绍: Functional studies dealing with identification, structure-function relationships or biological activity of: small regulatory RNAs (miRNAs, siRNAs and piRNAs) associated with the RNA interference pathway small nuclear RNAs, small nucleolar and tRNAs derived small RNAs other types of small RNAs, such as those associated with splice junctions and transcription start sites long non-coding RNAs, including antisense RNAs, long ''intergenic'' RNAs, intronic RNAs and ''enhancer'' RNAs other classes of RNAs such as vault RNAs, scaRNAs, circular RNAs, 7SL RNAs, telomeric and centromeric RNAs regulatory functions of mRNAs and UTR-derived RNAs catalytic and allosteric (riboswitch) RNAs viral, transposon and repeat-derived RNAs bacterial regulatory RNAs, including CRISPR RNAS Analysis of RNA processing, RNA binding proteins, RNA signaling and RNA interaction pathways: DICER AGO, PIWI and PIWI-like proteins other classes of RNA binding and RNA transport proteins RNA interactions with chromatin-modifying complexes RNA interactions with DNA and other RNAs the role of RNA in the formation and function of specialized subnuclear organelles and other aspects of cell biology intercellular and intergenerational RNA signaling RNA processing structure-function relationships in RNA complexes RNA analyses, informatics, tools and technologies: transcriptomic analyses and technologies development of tools and technologies for RNA biology and therapeutics Translational studies involving long and short non-coding RNAs: identification of biomarkers development of new therapies involving microRNAs and other ncRNAs clinical studies involving microRNAs and other ncRNAs.
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