Dual function for U2AF35 in AG-dependent pre-mRNA splicing
Dual function for U2AF35 in AG-dependent pre-mRNA splicing
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DOI：
10.1128/mcb.21.22.7673-7681.2001
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发表时间：
2001-11-01
影响因子：
5.3
通讯作者：
Valcárcel, J
中科院分区：
文献类型：
--
作者：
Guth, S;Tange, TO;Valcárcel, J
剪接因子U2 AF是高等真核生物中U2小核RNP向pre-mRNA募集所必需的。U2 AF的65-kDa亚基（U2 AF（65））与3'剪接位点之前的多聚嘧啶（Py）区结合，而35-kDa亚基（U2 AF（35））与内含子3'端的保守AG二核苷酸接触。已经表明，U2 AF 35和3'剪接位点AG之间的相互作用可以稳定U2 AF（65）与弱Py束的结合，所述弱Py束是所谓的AG依赖性前mRNA的特征。U2 AF（35）还涉及丝氨酸-丝氨酸（RS）结构域介导的与结合至外显子剪接增强子（ESE）的SR蛋白家族的剪接因子的桥接相互作用，并且这些相互作用还可以稳定U2 AF（65）结合。通过在寡聚（dT）-纤维素中的色谱法来补充耗尽U2 AF的核提取物的剪接活性，对于一些前体mRNA，仅需要U2 AF 65的存在。相反，小鼠免疫球蛋白M（IgM）M1-M2前mRNA的剪接需要两个U2 AF亚基。在本报告中，我们研究了序列元件（例如，Py束强度，3'剪接位点AG，ESE）负责IgM的U2 AF（35）依赖性。结果表明：（i）IgM底物是AG依赖性前体mRNA，（ii）U2 AF（35）依赖性与AG依赖性相关，（iii）外显子2第一个核苷酸的身份对U2 AF（35）功能很重要。相比之下，RS结构域介导的与结合于ESE的SR蛋白的相互作用似乎是不稳定的，因为外显子M2中存在的富含嘌呤的ESE对于U2 AF（35）活性不是必需的，并且因为仅由假RNA识别基序结构域组成并且缺乏RS结构域的U2 AF（35）的截短突变体在我们的互补测定中是有活性的。虽然U2 AF（35）的一些作用可以用增强的U2 AF（65）结合来解释，但U2 AF（35）的其他活性与U2 AF（65）与Py束的交联增加无关。总的来说，结果表明，除了稳定U2 AF（65）结合之外，U2 AF（35）与共有3'剪接位点的相互作用还触发剪接体组装中的事件，从而揭示了U2 AF（35）在前体mRNA剪接中的双重功能。
The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF(65)) binds to the polypyrimidine (Py) tract preceding the 3' splice site, while the 35-kDa subunit (U2AF(35)) contacts the conserved AG dinucleotide at the 3' end of the intron. It has been shown that the interaction between U2AF35 and the 3' splice site AG can stabilize U2AF(65) binding to weak Py tracts characteristic of so-called AG-dependent pre-mRNAs. U2AF(35) has also been implicated in arginine-serine (RS) domain-mediated bridging interactions with splicing factors of the SR protein family bound to exonic splicing enhancers (ESE), and these interactions can also stabilize U2AF(65) binding. Complementation of the splicing activity of nuclear extracts depleted of U2AF by chromatography in oligo(dT)-cellulose requires, for some pre-mRNAs, only the presence of U2AF65. In contrast, splicing of a mouse immunoglobulin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report we have investigated the sequence elements (e.g., Py tract strength, 3' splice site AG, ESE) responsible for the U2AF(35) dependence of IgM. The results indicate that (i) the IgM substrate is an AG-dependent pre-mRNA, (ii) U2AF(35) dependence correlates with AG dependence, and (iii) the identity of the first nucleotide of exon 2 is important for U2AF(35) function. In contrast, RS domain-mediated interactions with SR proteins bound to the ESE appear to be dispensable, because the purine-rich ESE present in exon M2 is not essential for U2AF(35) activity and because a truncation mutant of U2AF(35) consisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in our complementation assays. While some of the effects of U2AF(35) can be explained in terms of enhanced U2AF(65) binding, other activities of U2AF(35) do not correlate with increased cross-linking of U2AF(65) to the Py tract. Collectively, the results argue that interaction of U2AF(35) with a consensus 3' splice site triggers events in spliceosome assembly in addition to stabilizing U2AF(65) binding, thus revealing a dual function for U2AF(35) in pre-mRNA splicing.