Zhang L, Ashiya M, Sherman T G, Grabowski P J
Howard Hughes Medical Institute, University of Pittsburgh, Pennsylvania 15260, USA.
RNA. 1996 Jul;2(7):682-98.
Tissue-and stage-specific pre-mRNA splicing events are prevalent in mammals, yet molecular details are lacking about these important mechanisms of posttranscriptional gene control. In this study, we investigate the regulated splicing of rat gamma 2 pre-mRNA, a subunit of the GABAA receptor, as a step toward understanding the molecular basis of a neuron-specific splicing event involving cassette exon selection. Cell-and substrate-specific regulation of gamma 2 pre-mRNA is recapitulated in a neuronal cell line derived from the cerebellum, which produces enhanced levels of the exon-selected mRNA. In contrast, a control cell line derived from non-neuronal cells of the pituitary produces prominent levels of the unregulated, exon-skipped mRNA. The cerebellar and pituitary cell lines are well matched in overall splicing efficiency and produce an invariant pattern of splicing for a control substrate, which is alternatively spliced but not regulated in this system. The appropriateness of the two cell lines is indicated by an extended mRNA mapping experiment, which documents the region-specific switch in exon selection throughout rat brain. Using this pair of cell lines, we show that large intron segments flanking the regulated exon are dispensable for regulation. These intron regions have been deleted to generate a minimal splicing substrate for the purpose of identifying essential RNA elements. In this context, we show that essential nucleotides are located at positions +7, +8, and +9 of the regulated exon and in a 9-nt adenosine-rich region of the adjacent 3' splice site. Due to the proximity and base complementarity of the required nucleotides, experiments were devised to test models involving the recognition of two single-stranded signals, or one duplex RNA signal. These results clearly disfavor the duplex RNA recognition model and indicate that the required regions are recognized as independent, single strands in neuronal cells. A weak 5' splice site adjacent to the regulated exon is required as a third essential element. Although the importance of a weak 5' splice site is common to other regulated systems such as NCAM, the essential nucleotides in the exon and 3' splice site region defined in this study for gamma 2 splicing regulation are novel.
组织和阶段特异性的前体mRNA剪接事件在哺乳动物中普遍存在，但对于这些转录后基因调控的重要机制，分子细节仍不清楚。在本研究中，我们研究大鼠γ2前体mRNA（一种GABAA受体亚基）的调控剪接，作为理解涉及盒式外显子选择的神经元特异性剪接事件分子基础的一个步骤。γ2前体mRNA的细胞和底物特异性调控在源自小脑的神经元细胞系中得以重现，该细胞系产生增强水平的外显子选择mRNA。相比之下，源自垂体非神经元细胞的对照细胞系产生大量未受调控的、外显子跳跃的mRNA。小脑和垂体细胞系在整体剪接效率上匹配良好，并且对于对照底物产生不变的剪接模式，该对照底物在此系统中是可变剪接但不受调控的。一个扩展的mRNA图谱实验表明了这两种细胞系的适用性，该实验记录了大鼠脑中整个外显子选择的区域特异性转换。使用这对细胞系，我们表明受调控外显子两侧的大内含子片段对于调控是可有可无的。为了鉴定必需的RNA元件，已删除这些内含子区域以产生最小剪接底物。在这种情况下，我们表明必需核苷酸位于受调控外显子的+7、+8和+9位以及相邻3'剪接位点的一个富含腺苷的9核苷酸区域。由于所需核苷酸的接近性和碱基互补性，设计了实验来测试涉及识别两个单链信号或一个双链RNA信号的模型。这些结果明显不支持双链RNA识别模型，并表明所需区域在神经元细胞中被识别为独立的单链。靠近受调控外显子的一个弱5'剪接位点是第三个必需元件。尽管弱5'剪接位点的重要性在其他调控系统如神经细胞黏附分子（NCAM）中是常见的，但本研究中为γ2剪接调控定义的外显子和3'剪接位点区域中的必需核苷酸是新发现的。
