CUGBP2 directly interacts with U2 17S snRNP components and promotes U2 snRNA binding to cardiac troponin T pre-mRNA.
CUGBP2 directly interacts with U2 17S snRNP components and promotes U2 snRNA binding to cardiac troponin T pre-mRNA.
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DOI：
10.1093/nar/gkp346
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发表时间：
2009-07
影响因子：
14.9
通讯作者：
Cooper TA
中科院分区：
文献类型：
--
作者：
Goo YH;Cooper TA
CUGBP2 （etr3 /NAPOR/BRUNOL3）通过结合下游内含子的第21位和第74位，促进心肌肌钙蛋白T （cTNT）外显子5的包含。CUGBP2激活cTNT外显子5包合的分子机制尚不清楚。我们的研究结果表明，CUGBP2通过一种新机制促进外显子的内含，其中CUGBP2直接与活化的U2 snRNP的组分相互作用，并增强U2 snRNP与位于外显子上游分支位点的结合。通过体外剪接实验，我们发现重组CUGBP2增强了cTNT前mrna复合物A的形成。复合物A的增强组装需要上下游的内含子都符合cugbp2的下游结合位点和U2 snRNP结合的上游分支位点的双重要求。我们还发现CUGBP2增强了U2 snRNA与cTNT前mrna的结合，这与增强的复合物A组装一致。利用串联亲和纯化纯化CUGBP2相互作用蛋白，证明了核心17S U2 snRNP成分SF3b145和SF3b49直接与CUGBP2结合。我们得出结论，CUGBP2通过与17S snRNP复合物的组分形成直接相互作用来激活外显子包合，并招募和/或稳定U2 snRNP的结合。
CUGBP2 (ETR-3/NAPOR/BRUNOL3) promotes inclusion of cardiac troponin T (cTNT) exon 5 via binding between positions 21 and 74 of the downstream intron. The molecular mechanism by which CUGBP2 activates cTNT exon 5 inclusion is unknown. Our results suggest that CUGBP2 promotes exon inclusion by a novel mechanism in which CUGBP2 directly interacts with components of the activated U2 snRNP and enhances binding of U2 snRNP to the branch site located upstream of the exon. Using an in vitro splicing assay, we show that recombinant CUGBP2 enhances complex A formation of a cTNT pre-mRNA. Enhanced complex A assembly requires both the upstream and downstream introns consistent with dual requirements for the downstream CUGBP2-binding site and an upstream branch site for U2 snRNP binding. We also show that CUGBP2 enhances binding of U2 snRNA to the cTNT pre-mRNA consistent with enhanced complex A assembly. Purification of CUGBP2-interacting proteins using tandem affinity purification leads to the demonstration that the core 17S U2 snRNP components, SF3b145 and SF3b49 bind directly to CUGBP2. We conclude that CUGBP2 activates exon inclusion by forming direct interactions with components of the 17S snRNP complex and recruits and/or stabilizes binding of U2 snRNP.
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