Differential recruitment of pre-mRNA splicing factors to alternatively spliced transcripts in vivo.
Differential recruitment of pre-mRNA splicing factors to alternatively spliced transcripts in vivo.
复制标题
将前MRNA剪接因子的差异募集到体内剪接的转录本。
DOI：
10.1371/journal.pbio.0030374
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发表时间：
2005-11
期刊：
影响因子：
9.8
通讯作者：
Misteli, Tom
中科院分区：
文献类型：
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作者：
Mabon, Stephen A;Misteli, Tom
哺乳动物细胞中的选择性剪接已被认为在很大程度上受基础剪接因子与前体mRNA模板的组合结合控制。该模型预测不同的前mRNA剪接因子与选择性剪接的转录本相关。然而，没有实验证据的差异招募剪接因子转录不同的剪接命运。在这里，我们使用定量单细胞成像来测试体内的这一关键预测。我们发现，不同的剪接因子的组合被招募到完整细胞中的选择性剪接转录的位点。当包含外显子10时，丝氨酸/精氨酸蛋白剪接因子的子集（包括SF 2/ASF、SC 35和SRp20）被有效地募集到tau基因，而当排除外显子10时，这些因子不太频繁地与tau转录位点相关。相反，其他几种剪接因子的募集频率与剪接结果无关。SF2/ASF的突变分析表明，差异募集需要蛋白质-蛋白质以及蛋白质-RNA相互作用。各种剪接因子的差异行为为前体mRNA的组合占据提供了基础。这些观察结果代表了前mRNA剪接因子与可变剪接转录本差异相关的第一个体内证据。他们证实了选择性剪接随机模型的关键预测，其中通用前mRNA剪接因子的不同组合集有助于剪接结果。定量单细胞成像揭示了不同的剪接因子的组合招募到网站的选择性剪接转录在完整的细胞。
Alternative splicing in mammalian cells has been suggested to be largely controlled by combinatorial binding of basal splicing factors to pre-mRNA templates. This model predicts that distinct sets of pre-mRNA splicing factors are associated with alternatively spliced transcripts. However, no experimental evidence for differential recruitment of splicing factors to transcripts with distinct splicing fates is available. Here we have used quantitative single-cell imaging to test this key prediction in vivo. We show that distinct combinations of splicing factors are recruited to sites of alternatively spliced transcripts in intact cells. While a subset of serine/arginine protein splicing factors, including SF2/ASF, SC35, and SRp20, is efficiently recruited to the tau gene when exon 10 is included, these factors are less frequently associated with tau transcription sites when exon 10 is excluded. In contrast, the frequency of recruitment of several other splicing factors is independent of splicing outcome. Mutation analysis of SF2/ASF shows that both protein–protein as well as protein–RNA interactions are required for differential recruitment. The differential behavior of the various splicing factors provides the basis for combinatorial occupancy at pre-mRNAs. These observations represent the first in vivo evidence for differential association of pre-mRNA splicing factors with alternatively spliced transcripts. They confirm a key prediction of a stochastic model of alternative splicing, in which distinct combinatorial sets of generic pre-mRNA splicing factors contribute to splicing outcome. Quantitative single-cell imaging reveals distinct combinations of splicing factors recruited to sites of alternatively spliced transcripts in intact cells.