While RNAs are known to misfold, the underlying molecular causes remain unclear, and focused on alternative secondary structures. Conversely, how RNA chaperones function in a biological context to promote folding beyond duplex annealing, remains unknown. Here we show in a combination of DMS-MaPseq, structural analyses, biochemical experiments, and yeast genetics that three-way junctions are prone to misfolding during assembly of the small ribosomal subunit in vivo. We identify ubiquitous roles for ribosome assembly factors in chaperoning their folding by preventing the formation of tertiary interactions. In the absence of these assembly factors, tertiary interactions kinetically trap misfolded three-way junctions, thereby blocking further progress in the assembly cascade. While these protein chaperones act indirectly by binding the interaction partners, our analyses also suggest direct roles for snoRNAs in binding and chaperoning three-way junctions during transcription. This work furthermore shows that the dissociation of assembly factors renders reversible folding steps irreversible, thereby setting up a timer that regulates not just the flux of assembly but also dictates the propensity of misfolded intermediates to escape quality control. Finally, the data demonstrate how RNA chaperones act locally to unfold specific tertiary interactions, in contrast to protein chaperones, which globally unfold misfolded proteins.

中文翻译：

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组装因子伴侣rRNA通过分离容易错折叠的螺旋连接折叠

尽管已知RNA会错折叠，但潜在的分子原因仍不清楚，并集中在其他二级结构上。相反，RNA伴侣如何在生物学环境中发挥作用以促进折叠超出双链退火，仍然未知。在这里，我们结合DMS-MaPseq，结构分析，生化实验和酵母遗传学表明，在体内组装小核糖体亚基的过程中，三向连接易于错折叠。我们通过阻止三级相互作用的形成来确定核糖体装配因子在伴侣折叠中的普遍作用。在没有这些装配因素的情况下，三级相互作用在动力学上捕获了错误折叠的三通接头，从而阻碍了装配级联的进一步进展。虽然这些蛋白伴侣通过结合相互作用伴侣而间接发挥作用，但我们的分析还表明，snoRNA在转录过程中结合和伴侣三向连接中具有直接作用。这项工作进一步表明，组装因子的分离使得可逆的折叠步骤不可逆，从而建立了一个计时器，该计时器不仅调节组装的流量，而且还指示了错折叠的中间体逃避质量控制的可能性。最后，数据证明了RNA分子伴侣如何在局部发挥作用，以展开特定的三级相互作用，与蛋白质伴侣蛋白相反，后者可以整体展开错折叠的蛋白质。这项工作进一步表明，组装因子的分离使得可逆的折叠步骤不可逆，从而建立了一个计时器，该计时器不仅调节组装的流量，而且还指示了错折叠的中间体逃避质量控制的可能性。最后，数据证明了RNA分子伴侣如何在局部发挥作用，以展开特定的三级相互作用，与蛋白质伴侣蛋白相反，后者可以整体展开错折叠的蛋白质。这项工作进一步表明，组装因子的分离使得可逆的折叠步骤不可逆，从而建立了一个计时器，该计时器不仅调节组装的流量，而且还指示了错折叠的中间体逃避质量控制的可能性。最后，数据证明了RNA分子伴侣如何在局部发挥作用，以展开特定的三级相互作用，与蛋白质伴侣蛋白相反，后者可以整体展开错折叠的蛋白质。