Rino José, Desterro Joana M P, Pacheco Teresa R, Gadella Theodorus W J, Carmo-Fonseca Maria
Instituto de Medicina Molecular, Faculdade de Medicina, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal.
Mol Cell Biol. 2008 May;28(9):3045-57. doi: 10.1128/MCB.02015-07. Epub 2008 Feb 19.
Splicing factors SF1 and U2AF associate cooperatively with pre-mRNA and play a crucial role in 3' splice site recognition during early steps of spliceosome assembly. Formation of the active spliceosome subsequently displaces SF1 in a remodeling process that stabilizes the association of U2 snRNP with pre-mRNA. Fluorescence microscopy shows SF1 and U2AF distributed throughout the nucleoplasm, where transcription occurs, with additional concentration in nuclear speckles, where splicing factors accumulate when not engaged in splicing. Fluorescence recovery after photobleaching analysis in live cells shows that the mobilities of SF1 and the two subunits of U2AF (U2AF(65) and U2AF(35)) are correlated with the abilities of these proteins to interact with each other. Direct binding of SF1 to U2AF(65) was demonstrated by fluorescence resonance energy transfer in both the nucleoplasm and nuclear speckles. This interaction persisted after transcription inhibition, suggesting that SF1 associates with U2AF in a splicing-independent manner. We propose that SF1 and U2AF form extraspliceosomal complexes before and after taking part in the assembly of catalytic spliceosomes.
剪接因子SF1和U2AF与前体mRNA协同结合，并在剪接体组装的早期步骤中对3'剪接位点识别起关键作用。活性剪接体的形成随后在一个重塑过程中取代SF1，该过程稳定了U2 snRNP与前体mRNA的结合。荧光显微镜显示SF1和U2AF分布于整个发生转录的核质中，在核斑中也有额外的聚集，核斑是剪接因子在不参与剪接时聚集的地方。活细胞中的光漂白后荧光恢复分析表明，SF1和U2AF的两个亚基（U2AF(65)和U2AF(35)）的迁移率与这些蛋白质相互作用的能力相关。通过荧光共振能量转移在核质和核斑中均证实了SF1与U2AF(65)的直接结合。转录抑制后这种相互作用仍然存在，表明SF1以不依赖剪接的方式与U2AF结合。我们提出，SF1和U2AF在参与催化剪接体组装之前和之后形成额外的剪接体外复合物。
