mRNA secondary structures fold sequentially but exchange rapidly in vivo.
mRNA secondary structures fold sequentially but exchange rapidly in vivo.
复制标题
DOI：
10.1371/journal.pbio.1000307
复制
发表时间：
2010-02-09
期刊：
影响因子：
9.8
通讯作者：
Fedor MJ
中科院分区：
文献类型：
--
作者：
Mahen EM;Watson PY;Cottrell JW;Fedor MJ
RNA折叠的自切割分析表明，mRNA结构在体外和体内按顺序折叠，但相邻结构之间的交换在体内比在体外快得多。 RNA 采用确定的结构来执行生物活性，而替代结构之间的构象转变对于几乎所有 RNA 介导的过程都至关重要，从细菌核糖开关的代谢物激活到真核生物中的前 mRNA 剪接和病毒复制。在生物学背景下对 RNA 折叠反应进行机制分析具有挑战性，因为功能性 RNA 结构的组装和生物功能的执行之间通常会介入许多步骤。我们开发了一种系统，利用自裂解来直接在体内探测二级结构折叠和交换的机制，以监测促进或抑制核酶组装的互斥结构之间的竞争。在之前的工作中，上游结构比下游结构在体外转录过程中阻断核酶组装更有效，这与顺序折叠机制一致。然而，上游和下游结构在体内同样良好地阻断核酶组装，这表明细胞内折叠结果反映了热力学平衡或连续序列的退火在动力学上是有利的。我们扩展了这些研究，以了解热力学稳定性何时（如果有的话）成为体内替代 RNA 结构之间快速平衡的障碍。我们发现，狭窄的热力学阈值决定了体内 RNA 折叠结果是由动力学还是热力学控制。 mRNA二级结构在体内按顺序折叠，但相邻二级结构之间的交换在体内比在体外快得多。先前的研究表明，简单的碱基配对 RNA 螺旋在体内和体外以相似的速率解离，因此相邻结构之间的交换必须通过不同的机制发生，该机制可能涉及与新生转录物相关的蛋白质促进分支迁移。正确折叠的 RNA 对于几乎所有 RNA 介导的过程（从基因表达的反馈调节到 RNA 成熟）都至关重要。 RNA 在活细胞中采用特定结构的能力是非凡的，因为它们在体外容易被困在稳定的错误折叠结构的混合物中。使用插入核酶和自切割的 mRNA 来监测互斥结构之间的竞争，我们之前表明，在体外 RNA 合成过程中，上游结构主导折叠结果，表明折叠是顺序发生的。然而，当在体内研究时，上游和下游结构在酵母中同样有效地阻断核酶组装，提供了细胞内折叠结果反映替代结构的相对稳定性的证据。我们发现，即使下游结构更稳定，非常稳定的上游结构也可以阻止下游结构在体内的组装，并且狭窄的稳定性阈值决定了折叠和展开速率或热力学稳定性是否控制折叠结果。因此，mRNA在体外和体内顺序折叠，但相邻结构之间的交换在体内比在体外更快。简单的RNA结构在体内和体外以相似的速率展开，因此体内相邻结构之间的交换可能通过一种独特的、逐步的机制发生，该机制可以通过与新生RNA相关的蛋白质来促进。
Self-cleavage assays of RNA folding reveal that mRNA structures fold sequentially in vitro and in vivo, but exchange between adjacent structures is much faster in vivo than it is in vitro. RNAs adopt defined structures to perform biological activities, and conformational transitions among alternative structures are critical to virtually all RNA-mediated processes ranging from metabolite-activation of bacterial riboswitches to pre-mRNA splicing and viral replication in eukaryotes. Mechanistic analysis of an RNA folding reaction in a biological context is challenging because many steps usually intervene between assembly of a functional RNA structure and execution of a biological function. We developed a system to probe mechanisms of secondary structure folding and exchange directly in vivo using self-cleavage to monitor competition between mutually exclusive structures that promote or inhibit ribozyme assembly. In previous work, upstream structures were more effective than downstream structures in blocking ribozyme assembly during transcription in vitro, consistent with a sequential folding mechanism. However, upstream and downstream structures blocked ribozyme assembly equally well in vivo, suggesting that intracellular folding outcomes reflect thermodynamic equilibration or that annealing of contiguous sequences is favored kinetically. We have extended these studies to learn when, if ever, thermodynamic stability becomes an impediment to rapid equilibration among alternative RNA structures in vivo. We find that a narrow thermodynamic threshold determines whether kinetics or thermodynamics govern RNA folding outcomes in vivo. mRNA secondary structures fold sequentially in vivo, but exchange between adjacent secondary structures is much faster in vivo than it is in vitro. Previous work showed that simple base-paired RNA helices dissociate at similar rates in vivo and in vitro so exchange between adjacent structures must occur through a different mechanism, one that likely involves facilitation of branch migration by proteins associated with nascent transcripts. Properly folded RNAs are critical for virtually all RNA-mediated processes ranging from feedback regulation of gene expression to RNA maturation. The ability of RNAs to adopt specific structures in living cells is remarkable given their propensity to become trapped in a mixture of stable, misfolded structures in vitro. Using mRNA with an inserted ribozyme and self-cleavage to monitor competition between mutually exclusive structures, we previously showed that upstream structures dominated folding outcomes during RNA synthesis in vitro, suggesting that folding occurs sequentially. However, when studied in vivo upstream and downstream structures blocked ribozyme assembly equally well in yeast, providing evidence that intracellular folding outcomes reflect the relative stability of alternative structures. We find that very stable upstream structures can block assembly of downstream structures in vivo even when the downstream structures are more stable, and that a narrow threshold of stability determines whether folding and unfolding rates or thermodynamic stability govern folding outcomes. Thus, mRNAs fold sequentially in vitro and in vivo but exchange between adjacent structures is faster in vivo than in vitro. Simple RNA structures unfold at similar rates in vivo and in vitro, so exchange between adjacent structures in vivo probably occurs through a distinct, step-wise mechanism that could be facilitated by proteins associated with nascent RNAs.
登录
查看更多内容
影响因子：
15
作者：
Gluick, TC;Yadav, S
通讯作者：
Yadav, S
使用基于结构的比对，在人和小鼠mRNA的未翻译区域中挖掘小的RNA结构元素。
影响因子：
4.4
作者：
Khaladkar M;Liu J;Wen D;Wang JT;Tian B
通讯作者：
Tian B
影响因子：
5.6
作者：
GRALLA, J;CROTHERS, DM
通讯作者：
CROTHERS, DM
影响因子：
5.6
作者：
Diegelman-Parente, A;Bevilacqua, PC
通讯作者：
Bevilacqua, PC
影响因子：
5.6
作者：
Donahue, CP;Yadava, RS;Fedor, MJ
通讯作者：
Fedor, MJ