Mutations in the SF1-U2AF59-U2AF23 complex cause exon skipping in schizosaccharomyces pombe
Mutations in the SF1-U2AF59-U2AF23 complex cause exon skipping in schizosaccharomyces pombe
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DOI：
10.1074/jbc.m609430200
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发表时间：
2007-01-26
影响因子：
4.8
通讯作者：
Tani, Tokio
中科院分区：
文献类型：
--
作者：
Haraguchi, Noriko;Andoh, Tomoko;Tani, Tokio
为了鉴定参与确保组成型剪接前mRNA中5'至3'外显子有序连接机制的基因，我们使用报告质粒筛选了引起裂殖酵母粟酒裂殖酵母中外显子跳跃的突变体，该报告质粒包含ura 4（+）基因和nda 3内含子1-外显子2-内含子2序列。报告质粒被设计为当在剪接反应期间中心nda 3外显子被跳过时产生功能性ura 4（+）mRNA。我们通过紫外线照射诱变携带质粒的细胞，并分离出34个ura（+）突变体，这些突变体在基本培养基上生长。其中，8个突变体被发现是温度敏感（ts）的增长。互补分析表明，ts突变体属于三个不同的互补组命名ods（有序剪接）1，2和3。RT-PCR分析表明，ods突变体中确实产生了外显子跳跃产物。我们克隆了引起ods突变的基因，发现ods 1（+）、ods 2（+）和ods 3（+）分别编码剪接因子Prp 2 p/U2 AF（59）、U2 AF（23）和SF 1，它们形成SF 1-U2 AF（59）-U2 AF 23复合物，参与识别前体mRNA中的分支点和3'剪接位点序列。我们还发现，报告质粒中SF 1-U2 AF（59）-U2 AF（23）结合序列的突变导致野生型S.粟酒裂殖酵母细胞此外，降低转录延伸率的药物被发现可以抑制ods突变体中的外显子跳跃。这些结果表明，SF 1-U2 AF（59）-U2 AF 23复合物对新生前mRNA的共转录识别对于S.粟酒
To identify genes involved in the mechanism to ensure ordered 5' to 3' exon joining in constitutively spliced pre-mRNAs, we screened for mutants that cause exon skipping in the fission yeast Schizosaccharomyces pombe using a reporter plasmid, which contains the ura4(+) gene with the nda3 intron 1-exon 2-intron 2 sequence. The reporter plasmid was designed to produce the functional ura4(+) mRNA, when the central nda3 exon is skipped during the splicing reaction. We mutagenized cells harboring the plasmid by UV irradiation and isolated 34 ura(+) mutants that grew on minimal medium. Of those, eight mutants were found to be temperature sensitive (ts) for growth. Complementation analyses revealed that the ts mutants belong to three distinct complementation groups named ods (ordered splicing) 1, 2, and 3. RT-PCR analyses showed that products of exon skipping were actually generated in the ods mutants. We cloned the genes responsible for the ods mutations, and found that ods1(+), ods2(+), and ods3(+) encode splicing factors Prp2p/ U2AF(59), U2AF(23), and SF1, respectively, which form a SF1-U2AF(59)-U2AF23 complex involved in recognition of the branchpoint and 3' splice site sequences in a pre-mRNA. We also showed that mutations in the SF1-U2AF(59)-U2AF(23) binding sequences in the reporter plasmid result in exon skipping in wild-type S. pombe cells. In addition, drugs that decrease the rate of transcription elongation were found to suppress the exon skipping in the ods mutants. These results suggest that co-transcriptional recognition of a nascent pre-mRNA by the SF1-U2AF(59)- U2AF23 complex is essential for ordered exon joining in constitutive splicing in S. pombe.