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Author:
Date:
2015-04-20
[Abstract] microRNAs (miRNAs) are small non-coding RNAs, regulating most if not all, biological processes in eukaryotic organisms. miRNAs are initially processed from primary transcripts (pri-miRNAs) to produce miRNA precursors (pre-miRNAs), that are further processed into miRNA and its complementary strands (miRNA/*). In Arabidopsis, and possibly other plants, the processing from pri-miRNAs to pre-miRNAs and from pre-miRNAs to miRNA/* are both implemented through Dicer-like 1 (DCL1) complexes. Recently, we demonstrated isolation of DCL1 complexes of unprecedented quality from in planta. We further successfully reconstituted DCL1 cleavage assays in vitro that were able to fully recapitulate in vivo miRNA biogenesis. Here we provide a detailed protocol of DCL1 ...
[摘要] 微小RNA（miRNA）是小的非编码RNA，其调节真核生物中的大多数（如果不是全部）生物过程。 miRNA最初从初级转录物（pri-miRNA）加工以产生miRNA前体（前-miRNA），其进一步加工成miRNA及其互补链（miRNA/*）。在拟南芥和可能的其他植物中，从pri-miRNA到pre-miRNA和从pre-miRNA到miRNA/*的加工都通过Dicer样1（DCL1）复合物实现。最近，我们证明了从植物中以前的质量的DCL1复合物的分离。我们进一步成功地重建了能够完全重现体内 miRNA生物发生的DCL1切割测定。在这里我们提供DCL1重建测定的详细协议。该方案包括三个主要部分（图1）：1）pri-miRNA和pre-miRNA转录物的制备（方法A-C）; 2）通过免疫沉淀（IP）纯化来自本塞姆氏烟草（本塞姆氏烟草）的重组拟南芥DCL1机器（程序D和E）;和3）使用分离的DCL1复合物（步骤F）在放射性同位素标记的pri-miRNA或pre-miRNA的体外处理。这是我们的愿望，协议是RNAi社区研究机械问题或开发RNA沉默技术的强大工具。
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Author:
Date:
2014-09-20
[Abstract] This protocol describes the coupling of (i) “live” in vitro RNA transcription with (ii) binding by a radiolabeled, pre-formed tRNA followed by native gel electrophoresis and phosphorimager scan to visualize the complex. The necessity arose from the stable structure that one RNA forms in the absence of its interaction partner. The T-box leader RNA, a transcription control system, folds into a thermodynamically very stable stem-loop structure without the tRNA present, which makes in vitro binding interaction of both pre-formed RNAs very difficult. I therefore adjusted the binding assay to mimic the “natural” situation in the bacterial cell, where the pre-formed, stable tRNA is already present while the T-box leader RNA is actively transcribed by the RNA polymerase. The ...
[摘要] 该方案描述了（i）"活"体外RNA转录与（ii）通过放射性标记的预先形成的tRNA的结合，然后是天然凝胶电泳和磷光成像仪扫描以显现复合物的偶联。 必要性来自一种RNA在不存在其相互作用配偶体时形成的稳定结构。 T盒前导RNA，转录控制系统，折叠成热力学非常稳定的茎 - 环结构，没有tRNA存在，这使得体外结合两个预先形成的RNA的相互作用非常困难。 因此，我调整结合测定以模拟细菌细胞中的"天然"情况，其中预先形成的稳定的tRNA已经存在，而T盒前导RNA被RNA聚合酶主动转录。 方案的第一部分还描述了体外转录和tRNA的标记。
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