CONTROL OF ALTERNATIVE SPLICING BY THE DIFFERENTIAL BINDING OF U1 SMALL NUCLEAR RIBONUCLEOPROTEIN PARTICLE
CONTROL OF ALTERNATIVE SPLICING BY THE DIFFERENTIAL BINDING OF U1 SMALL NUCLEAR RIBONUCLEOPROTEIN PARTICLE
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DOI：
10.1126/science.1825520
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发表时间：
1991-03-01
期刊：
影响因子：
56.9
通讯作者：
GRABOWSKI, PJ
中科院分区：
文献类型：
--
作者：
KUO, HC;NASIM, FUH;GRABOWSKI, PJ
控制前体信使RNA选择性剪接的细胞因素在很大程度上是未知的，尽管这个过程在确定真核细胞中蛋白质的多样性方面发挥着核心作用。为了鉴定这些因子，使用了大鼠前原速激肽基因的片段，其中神经肽-γ和K的差异表达依赖于第四外显子(E4)的可变剪接。创建了三外显子片段 (E3-E4-E5) 的序列变体，从而对介导 E4 跳跃和 E4 包含之间剪接转换的因素进行了灵敏的测定。 E4 5'剪接位点的二核苷酸突变增加了该位点与U1小核RNA的碱基配对，导致E4的一致选择，而破坏碱基配对的对照突变导致一致的E4跳跃。在这些功能不同的底物上形成的剪接体的亲和力选择表明，剪接的极端差异是由 U1 小核核糖核蛋白颗粒 (snRNP) 与 E4 5' 剪接位点的差异结合介导的。这些数据表明，除了在选择 5' 剪接位点方面的既定作用之外，U1 snRNP 在 3' 外显子选择中也发挥着重要作用，并提供了对选择性剪接的可能机制的见解。
Cellular factors controlling alternative splicing of precursor messenger RNA are largely unknown, even though this process plays a central role in specifying the diversity of proteins in the eukaryotic cell. For the identification of such factors, a segment of the rat preprotachykinin gene was used in which differential expression of neuropeptides-gamma and K is dependent on alternative splicing of the fourth exon (E4). Sequence variants of the three-exon segment, (E3-E4-E5) were created, resulting in a sensitive assay for factors mediating the splicing switch between E4-skipping and E4-inclusion. A dinucleotide mutation in the 5' splice site of E4 that increases base-pairing of this site to U1 small nuclear RNA resulted in uniform selection of E4, whereas a control mutation that destroyed base-pairing resulted in uniform E4-skipping. Affinity selection of spliceosomes formed on these functionally distinct substrates revealed that the extreme difference in splicing was mediated by differential binding of the U1 small nuclear ribonucleoprotein particle (snRNP) to the 5' splice site of E4. These data show that, apart from its established role in selecting 5' splice sites, U1 snRNP plays a fundamental role in 3' exon selection and provides insight into possible mechanisms of alternative splicing.