Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.
Methods. 2021 Dec;196:30-35. doi: 10.1016/j.ymeth.2021.02.002. Epub 2021 Feb 10.
Circular RNAs (circRNAs) generated from back-splicing of exons have been found in a wide range of eukaryotic species and exert a variety of biological functions. Unlike canonical splicing, the mechanism of back-splicing has long remained elusive. We recently determined the cryo-EM structure of the yeast spliceosomal E complex assembled on introns, leading us to hypothesize that the same E complex can assemble across an exon forming the exon-definition complex. This complex, when assembled on long exons, goes through the splicing cycle and catalyzes back-splicing to generate circRNAs. Supporting this hypothesis, we purified the yeast post-catalytic spliceosomal P complex (the best complex in the splicing cycle to trap splicing products and intermediates) and detected canonical and back-splicing products as well as splicing intermediates. Here we describe in detail this procedure, which may be applied to other organisms to facilitate research on the biogenesis and regulation of circRNA.
环状 RNA（circRNAs）是通过外显子的反向剪接产生的，已在多种真核生物中发现，并发挥多种生物学功能。与经典剪接不同，反向剪接的机制长期以来一直难以捉摸。我们最近确定了在内含子上组装的酵母剪接体 E 复合物的冷冻电镜结构，这使我们假设相同的 E 复合物可以在形成外显子定义复合物的外显子上组装。当该复合物组装在长外显子上时，它会经历剪接循环，并催化反向剪接产生 circRNAs。支持这一假设，我们纯化了酵母催化后剪接体 P 复合物（剪接循环中捕获剪接产物和中间产物的最佳复合物），并检测到了经典剪接产物和反向剪接产物以及剪接中间产物。在这里，我们详细描述了这个过程，它可以应用于其他生物体，以促进 circRNA 生物发生和调控的研究。
