Flanking Sequence Cotranscriptionally Regulates Twister Ribozyme Activity
Flanking Sequence Cotranscriptionally Regulates Twister Ribozyme Activity
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DOI：
10.1021/acs.biochem.3c00506
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发表时间：
2023-12-22
期刊：
影响因子：
2.9
通讯作者：
Bevilacqua，Philip C.
中科院分区：
文献类型：
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作者：
McKinley，Lauren N.;Kern，Reuben G.;Bevilacqua，Philip C.
小的溶核核酶是切割它们自己的磷酸二酯骨架的RNA。虽然蛋白质酶受多种已知机制的调节，但核酶的调节方法仍不清楚。Twister是一类具有多种结构和催化性质的核酶。然而，很少有研究分析了扭转酶核酶在天然侧翼序列的背景下的活性，即使核酶在自然界中转录并不孤立存在。核酶与其相邻序列之间的相互作用可以诱导抑制自切割的构象变化，提供了一种可以自然地决定核酶在体内和合成应用中的活性的调节机制。到目前为止，已经在主要作物水稻（Oryza sativa）中鉴定出八种扭转酶核酶。在此，我们选择了几个扭转酶核酶从水稻和显示，他们的侧翼序列，使用已发表的RNA-seq数据集，结构探测，和共转录裂解测定的差异调节。我们发现Osa 1-2核酶不与其侧翼序列相互作用。然而，Osa 1-3和Osa 1-8核酶侧翼的序列形成减弱核酶自切割活性的无活性构象，在此称为“核酶原”。对于Osa 1-3核酶，我们表明，活性可以获救后，添加互补的反义寡核苷酸，这表明核酶可以通过外部信号控制。总之，我们的数据提供了一个合理的机制，其中侧翼序列差异调节核酶活性在体内。更广泛地说，局部调节核酶行为的能力在控制基因表达和合成生物学方面具有潜在的应用。
Small nucleolytic ribozymes are RNAs that cleave their own phosphodiester backbone. While proteinaceous enzymes are regulated by a variety of known mechanisms, methods of regulation for ribozymes remain unclear. Twister is one ribozyme class for which many structural and catalytic properties have been elucidated. However, few studies have analyzed the activity of twister ribozymes in the context of a native flanking sequence, even though ribozymes as transcribed in nature do not exist in isolation. Interactions between the ribozyme and its neighboring sequences can induce conformational changes that inhibit self-cleavage, providing a regulatory mechanism that could naturally determine ribozyme activityin vivoand in synthetic applications. To date, eight twister ribozymes have been identified within the staple crop rice (Oryza sativa). Herein, we select several twister ribozymes from rice and show that they are differentially regulated by their flanking sequence using published RNA-seq data sets, structure probing, and cotranscriptional cleavage assays. We found that the Osa 1–2 ribozyme does not interact with its flanking sequences. However, sequences flanking the Osa 1–3 and Osa 1–8 ribozymes form inactive conformations, referred to here as “ribozymogens”, that attenuate ribozyme self-cleavage activity. For the Osa 1–3 ribozyme, we show that activity can be rescued upon addition of a complementary antisense oligonucleotide, suggesting ribozymogens can be controlled via external signals. In all, our data provide a plausible mechanism wherein flanking sequence differentially regulates ribozyme activityin vivo. More broadly, the ability to regulate ribozyme behavior locally has potential applications in control of gene expression and synthetic biology.