Tsukahara T, Casciato C, Helfman D M
Cold Spring Harbor Laboratory, NY 11724.
Nucleic Acids Res. 1994 Jun 25;22(12):2318-25. doi: 10.1093/nar/22.12.2318.
We previously found that the splicing of exon 5 to exon 6 in the rat beta-TM gene required that exon 6 first be joined to the downstream common exon 8 (Helfman et al., Genes and Dev. 2, 1627-1638, 1988). Pre-mRNAs containing exon 5, intron 5 and exon 6 are not normally spliced in vitro. We have carried out a mutational analysis to determine which sequences in the pre-mRNA contribute to the inability of this precursor to be spliced in vitro. We found that mutations in two regions of the pre-mRNA led to activation of the 3'-splice site of exon 6, without first joining exon 6 to exon 8. First, introduction of a nine nucleotide poly U tract upstream of the 3'-splice site of exon 6 results in the splicing of exon 5 to exon 6 with as little as 35 nucleotides of exon 6. Second, introduction of a consensus 5'-splice site in exon 6 led to splicing of exon 5 to exon 6. Thus, three distinct elements can act independently to activate the use of the 3'-splice site of exon 6: (1) the sequences contained within exon 8 when joined to exon 6, (2) a poly U tract in intron 5, and (3) a consensus 5'-splice site in exon 6. Using biochemical assays, we have determined that these sequence elements interact with distinct cellular factors for 3'-splice site utilization. Although HeLa cell nuclear extracts were able to splice all three types of pre-mRNAs mentioned above, a cytoplasmic S100 fraction supplemented with SR proteins was unable to efficiently splice exon 5 to exon 6 using precursors in which exon 6 was joined to exon 8. We also studied how these elements contribute to alternative splice site selection using precursors containing the mutually exclusive, alternatively spliced cassette comprised of exons 5 through 8. Introduction of the poly U tract upstream of exon 6, and changing the 5'-splice site of exon 6 to a consensus sequence, either alone or in combination, facilitated the use of exon 6 in vitro, such that exon 6 was spliced more efficiently to exon 8. These data show that intron sequences upstream of an exon can contribute to the use of the downstream 5'-splice, and that sequences surrounding exon 6 can contribute to tissue-specific alternative splice site selection.
我们先前发现，大鼠β-珠蛋白基因中外显子5与外显子6的剪接要求外显子6首先与下游的共同外显子8连接（Helfman等人，《基因与发育》2，1627 - 1638，1988）。含有外显子5、内含子5和外显子6的前体mRNA在体外通常不进行剪接。我们进行了一项突变分析，以确定前体mRNA中的哪些序列导致该前体在体外无法进行剪接。我们发现，前体mRNA两个区域的突变导致外显子6的3'剪接位点激活，而无需先将外显子6与外显子8连接。首先，在外显子6的3'剪接位点上游引入一个九个核苷酸的聚U序列，导致外显子5与外显子6剪接，外显子6只需35个核苷酸。其次，在外显子6中引入一个共有5'剪接位点导致外显子5与外显子6剪接。因此，三种不同的元件可独立作用以激活外显子6的3'剪接位点的使用：（1）与外显子6连接时外显子8中包含的序列，（2）内含子5中的聚U序列，以及（3）外显子6中的共有5'剪接位点。通过生化分析，我们确定这些序列元件与不同的细胞因子相互作用以实现3'剪接位点的利用。虽然HeLa细胞核提取物能够剪接上述所有三种类型的前体mRNA，但补充了SR蛋白的细胞质S100组分无法使用外显子6与外显子8连接的前体有效地将外显子5与外显子6剪接。我们还使用含有由外显子5至8组成的相互排斥、可变剪接盒的前体研究了这些元件如何有助于可变剪接位点的选择。在外显子6上游引入聚U序列，以及将外显子6的5'剪接位点改变为共有序列，单独或组合使用，均有助于外显子6在体外的使用，使得外显子6更有效地与外显子8剪接。这些数据表明，外显子上游的内含子序列可有助于下游5'剪接的使用，并且外显子6周围的序列可有助于组织特异性可变剪接位点的选择。
