Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5' splice site recognition.
Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5' splice site recognition.
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DOI：
10.7554/elife.04986
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发表时间：
2015-01-02
期刊：
影响因子：
7.7
通讯作者：
Nagai K
中科院分区：
文献类型：
--
作者：
Kondo Y;Oubridge C;van Roon AM;Nagai K
U1 snRNP结合于前体mRNA的5′外显子-内含子连接处，因此在前体mRNA剪接的早期阶段起关键作用。我们提出了两种工程U1亚结构的晶体结构，它们一起以原子分辨率揭示了U1 snRNP内蛋白质-蛋白质和RNA-蛋白质相互作用的几乎完整的网络，并显示了U1 snRNP如何识别前mRNA的5′剪接位点。U1-C的锌指与pre-mRNA和U1 snRNA的5′端之间的双链体相互作用。RNA双链体的结合通过U1-C和剪接点周围的RNA骨架之间的氢键和静电相互作用来稳定，但U1-C与前mRNA没有碱基特异性接触。该结构与RNA结合试验一起表明，U1 snRNP对5′-剪接位点核苷酸的选择主要通过与U1 snRNA的碱基配对来实现，而U1-C微调错配的5′-剪接位点的相对亲和力。DOI：http://dx.doi.org/10.7554/eLife.04986.001基因是由很长的DNA组成的。编码蛋白质的基因区域（称为外显子）被称为内含子的非编码DNA片段打断。为了从基因中产生蛋白质，DNA被“转录”以形成前mRNA分子，其中内含子必须在称为剪接的过程中被移除。然后，剩余的外显子连接在一起形成一个成熟的mRNA分子，其中包含构建蛋白质的指令。剪接过程中的错误可能导致许多疾病，如癌症。一种被称为剪接体的分子机器负责剪接前mRNA分子。它由五种不同的复合物组成，称为小核核糖核蛋白颗粒（snRNP），它们又由许多蛋白质和RNA分子组成。剪接体在每次剪接时都重新组装，这个组装过程的早期步骤涉及称为U1的snRNP与前mRNA中内含子的起始部分相互作用。然后这种相互作用刺激剪接体其余部分的组装。2009年，研究人员报道了U1 snRNP的结构，但该结构没有包含足够的细节来揭示snRNP如何识别内含子的开始。Kondo，Oubridge等人，包括一些参与2009年工作的研究人员，现在更详细地展示了人类版本U1 snRNP的晶体结构。不能获得完整的U1 snRNP分子的高质量晶体结构，因为snRNP中RNA分子的排列阻止了规则晶体的形成。Kondo，Oubridge等人设计了U1 snRNP的两个亚组分，每个亚组分都结晶良好，并确定了它们的结构。这表明U1 snRNP的各个部分之间的相互作用形成了一个复杂的网络。U1 snRNP中存在的一种蛋白质，称为U1-C，以前曾被报道能够识别内含子本身，而不需要完整的U1 snRNP。Kondo，Oubridge等人揭示了情况并非如此，并且U1-C不直接读取内含子RNA序列。相反，U1 snRNP能够找到内含子的开始，因为U1 RNA可以稳定地结合到这个位点。然而，U1-C蛋白可以调节这种结合的强度，以确保剪接体可以与各种内含子起始序列（或信号）一起操作。DOI：http://dx.doi.org/10.7554/eLife.04986.002网站
U1 snRNP binds to the 5′ exon-intron junction of pre-mRNA and thus plays a crucial role at an early stage of pre-mRNA splicing. We present two crystal structures of engineered U1 sub-structures, which together reveal at atomic resolution an almost complete network of protein–protein and RNA-protein interactions within U1 snRNP, and show how the 5′ splice site of pre-mRNA is recognised by U1 snRNP. The zinc-finger of U1-C interacts with the duplex between pre-mRNA and the 5′-end of U1 snRNA. The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone around the splice junction but U1-C makes no base-specific contacts with pre-mRNA. The structure, together with RNA binding assays, shows that the selection of 5′-splice site nucleotides by U1 snRNP is achieved predominantly through basepairing with U1 snRNA whilst U1-C fine-tunes relative affinities of mismatched 5′-splice sites. DOI: http://dx.doi.org/10.7554/eLife.04986.001 Genes are made up of long stretches of DNA. The regions of a gene that code for proteins (known as exons) are interrupted by stretches of non-coding DNA called introns. To produce proteins from a gene, the DNA is ‘transcribed’ to form pre-mRNA molecules, from which the introns must be removed in a process called splicing. The remaining exons are then joined together to form a mature mRNA molecule that contains the instructions to build a protein. Errors in the splicing process can lead to numerous diseases, such as cancer. A molecular machine known as a spliceosome is responsible for splicing the pre-mRNA molecules. This consists of five different complexes called small nuclear ribonucleoprotein particles (snRNPs), which are in turn made up from numerous proteins and RNA molecules. The spliceosome assembles anew every time it splices, and an early step in this assembly process involves the interaction of an snRNP called U1 with the start of an intron in the pre-mRNA. This interaction then stimulates the assembly of the rest of the spliceosome. In 2009, researchers reported the structure of the U1 snRNP, but the structure did not contain enough detail to reveal how the snRNP recognizes the start of an intron. Kondo, Oubridge et al., including some of the researchers involved in the 2009 work, now present the crystal structure of the human version of the U1 snRNP in more detail. High-quality crystal structures of the complete U1 snRNP molecule could not be obtained because the arrangement of the RNA molecules in the snRNP prevented a regular crystal from forming. Kondo, Oubridge et al. instead engineered two subcomponents of U1 snRNP that each crystallized well, and determined their structures. This revealed that the interactions between the various parts of the U1 snRNP form a complex network. A protein present in the U1 snRNP, known as U1-C, had previously been reported to be able to recognize introns on its own—without requiring the complete U1 snRNP. Kondo, Oubridge et al. reveal that this is not the case and that U1-C does not read the intron RNA sequence directly. Instead, U1 snRNP is able to find the start of the intron because the U1 RNA can stably bind to this site. The U1-C protein can however adjust the strength of this binding to ensure that the spliceosome can operate with a variety of intron start sequences (or signals). DOI: http://dx.doi.org/10.7554/eLife.04986.002