Nuclear protein TIA-1 regulates COL2A1 alternative splicing and interacts with precursor mRNA and genomic DNA
Nuclear protein TIA-1 regulates COL2A1 alternative splicing and interacts with precursor mRNA and genomic DNA
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DOI：
10.1074/jbc.m702717200
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发表时间：
2007-08-17
影响因子：
4.8
通讯作者：
Sandell, Linda J.
中科院分区：
文献类型：
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作者：
McAlinden, Audrey;Liang, Li;Sandell, Linda J.
RNA结合蛋白TIA-1(T细胞限制性胞内抗原-1)在转录后调控机制中发挥作用，包括前体mRNA(Pre-mRNA)选择性剪接和mRNA翻译。利用由II型前胶原基因(COL2A1)的一部分组成的微型基因，我们发现TIA-1与COL2A1内含子2中一个保守的富含AU的顺式元件相互作用，并调节外显子2的选择性剪接。这个独特的、高度保守的包含茎环二级结构的顺式元件先前在我们的实验室被发现是一个基本基序，它在软骨形成过程中控制发育调节的外显子2剪接开关(McAlinden，A.，Havlioglu，N.，Leung，L.，Davies，S.R.和Sandell，L.J.J.化学。280、32700-32711)。核糖核蛋白免疫沉淀实验证实内源性TIA-1与COL2A1前-mRNA中富含AU的顺式元件结合。重要的是，我们还表明TIA-1与同等的DNA序列相互作用，倾向于单链DNA而不是双链DNA。染色质免疫沉淀分析(包括额外的核糖核酸酶步骤)证实了体内的这种相互作用。竞争分析表明，TIA-1与DNA的亲和力明显高于与RNA的亲和力。最后，我们证明了在活跃的转录过程中，这种强烈的DNA-TIA-1相互作用可以被RNA聚合酶破坏。这表明TIA-1在DNA和RNA配体之间穿梭，在转录水平和前mRNA选择性剪接水平上共同调节COL2A1的表达，具有潜在的新颖的双重作用。
The RNA-binding protein TIA-1 (T-cell-restricted intracellular antigen-1) functions in regulating post-transcriptional mechanisms, including precursor mRNA (pre-mRNA) alternative splicing and mRNA translation. Utilizing a mini-gene consisting of part of the type II procollagen gene (COL2A1), we show that TIA-1 interacts with a conserved AU-rich cis element in COL2A1 intron 2 and modulates alternative splicing of exon 2. This unique, highly conserved cis element containing stem-loop secondary structure was previously identified in our laboratory as an essential motif that controls the developmentally regulated exon 2 splicing switch during chondrogenesis (McAlinden, A., Havlioglu, N., Liang, L., Davies, S. R., and Sandell, L. J. (2005) J. Biol. Chem. 280, 32700-32711). In vivo binding of endogenous TIA-1 to the AU-rich cis element in COL2A1 pre-mRNA was confirmed by the ribonucleoprotein immunoprecipitation assay. Importantly, we also show that TIA-1 interacts with the equivalent DNA sequence with a preference for single-stranded rather than double-stranded DNA. Chromatin immunoprecipitation assays (including an additional RNase step) confirmed this interaction in vivo. Competition assays showed that TIA-1 apparently binds with higher affinity to DNA than to RNA. Finally, we show that this strong DNA-TIA-1 interaction can be disrupted by an RNA polymerase during active transcription. This suggests a potentially novel, dual role for TIA-1 in shuttling between DNA and RNA ligands to coregulate COL2A1 expression at the level of transcription and pre-mRNA alternative splicing.