2016年7月22日，生命科学联合中心施一公研究组于《科学》（Science）杂志就剪接体的结构与机理研究发表两篇长文（Research Article），题目分别为《酵母剪接体激活状态3.5埃的结构》（Structure of a Yeast Activated Spliceosome at 3.5 A Resolution）和《第一步催化反应后的酵母剪接体3.4埃的结构》（Structure of a Yeast Catalytic Step I Spliceosome at 3.4 A Resolution），报道了酿酒酵母（Saccharomyces cerevisiae）剪接体激活和剪接反应催化过程中两个重要状态的剪接体复合物近原子分辨率的三维结构，阐明了剪接体的激活和催化机制，从而进一步揭示了前体信使RNA剪接反应（pre-mRNA splicing，以下简称RNA剪接）的分子机理。
RNA剪接是真核生物从DNA到蛋白质信息传递中心法则的关键一环。其主要执行者是一个极其复杂的分子机器——剪接体。通过剪接反应，前体信使RNA中数量、长度不等的内含子被剔除，剩下的外显子按照特异顺序连接起来从而形成成熟的信使RNA（mRNA），进一步在核糖体的催化下被翻译成蛋白质。RNA剪接的化学本质就是前体信使RNA经历两步转酯反应完成剪和接在两个关键步骤，而每一步都需要由剪接体催化完成。
剪接体是一个由大量蛋白因子介导、核酸（RNA）催化的金属核酶（protein-directed metalloribozyme）。在剪接反应过程中，组成剪接体的蛋白质-核酸复合物及剪接因子按照高度精确的顺序进行结合和解聚，并伴随大规模的结构重组，组装成一系列具有不同组分和构象的统称为剪接体的分子机器，根据它们在RNA剪接过程中的生化性质，这些剪接体又被人为区分为B、Bact、B*、C、P、ILS等若干状态。获取剪接体在激活及催化反应过程中不同状态的结构是最基础也是最富挑战性的结构生物学难题之一。2015年8月，施一公研究组率先突破，在世界上首次报道了裂殖酵母剪接体处于ILS状态的3.6埃高分辨率结构。
在最新发表的两篇《科学》论文中，施一公研究组进一步探索并优化了蛋白提纯方案，捕获了性质良好的酿酒酵母剪接体分别处于激活状态（activated spliceosome，又称为Bact complex）和第一步催化反应后（catalytic step I spliceosome，又称为C complex）的优质样品，并利用单颗粒冷冻电镜技术和高效的数据分类方法，重构出了总体分辨率分别为3.5和3.4埃的两个高分辨率冷冻电镜结构，并搭建了原子模型（图1，2）。这两个复合物近原子分辨率三维结构的解析，首次完整地展示了第一步转酯反应前后pre-mRNA和起催化作用的snRNA的反应状态，以及剪接体内部蛋白组分的组装情况。尤为值得一提的是，催化核心区域的分辨率达到了2.8至3.0埃，清晰的展示出剪接反应中心的结构信息，为解释剪接体对pre-mRNA splicing的催化机制提供了迄今最为清晰的关键证据。
如上两个结构与该研究组之前报道的ILS剪接体及2016年1月报道的3.8埃的酿酒酵母tri-snRNP结构的对比更为深刻的揭示了剪接体在pre-mRNA剪接反应过程中作为核酶催化完成两步转酯反应的本质，是RNA剪接研究领域的又一突破性进展。
Structure of a yeast activated spliceosome at 3.5 Å resolution
Pre–messenger RNA (pre-mRNA) splicing is carried out by the spliceosome, which undergoes an intricate assembly and activation process. Here, we report an atomic structure of an activated spliceosome (known as the Bact complex) from Saccharomyces cerevisiae, determined by cryo–electron microscopy at an average resolution of 3.52 angstroms. The final refined model contains U2 and U5 small nuclear ribonucleoprotein particles (snRNPs), U6 small nuclear RNA (snRNA), nineteen complex (NTC), NTC-related (NTR) protein, and a 71-nucleotide pre-mRNA molecule, which amount to 13,505 amino acids from 38 proteins and a combined molecular mass of about 1.6 megadaltons. The 5ʹ exon is anchored by loop I of U5 snRNA, whereas the 5ʹ splice site (5ʹSS) and the branch-point sequence (BPS) of the intron are specifically recognized by U6 and U2 snRNA, respectively. Except for coordination of the catalytic metal ions, the RNA elements at the catalytic cavity of Prp8 are mostly primed for catalysis. The catalytic latency is maintained by the SF3b complex, which encircles the BPS, and the splicing factors Cwc24 and Prp11, which shield the 5ʹ exon–5ʹSS junction. This structure, together with those determined earlier, outlines a molecular framework for the pre-mRNA splicing reaction.
原文链接：http://science.sciencemag.org/content/353/6302/904
Structure of a yeast catalytic step I spliceosome at 3.4 Å resolution
Abstract
Each cycle of pre-mRNA splicing, carried out by the spliceosome, comprises two sequential transesterification reactions, which result in the removal of an intron and joining of two exons. Here we report an atomic structure of a catalytic step I spliceosome (known as the C complex) from Saccharomyces cerevisiae, determined by cryo-electron microscopy at an average resolution of 3.4 Å. In the structure, the 2′-OH of the invariant adenine nucleotide in the branch point sequence (BPS) is covalently joined to the phosphate at the 5′-end of the 5′-splice site (5′SS), forming an intron lariat. The freed 5′-exon remains anchored to loop I of U5 small nuclear RNA (snRNA), and the 5′SS and BPS of the intron form duplexes with conserved U6 and U2 snRNA sequences, respectively. Specific placement of these RNA elements at the catalytic cavity of Prp8 is stabilized by 15 protein components, including Snu114 and the splicing factors Cwc21, Cwc22, Cwc25, and Yju2. These features, representing the conformation of the spliceosome after the first-step reaction, predict structural changes that are needed for the execution of the second-step transesterification reaction.
原文链接：http://science.sciencemag.org/content/early/2016/07/20/science.aag2235
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来源：生物谷，science
本期编辑：Biodog