Nishiyama Takayuki, Hatano Hiroshi, Kurosaka Masahiro, Bolander Mark E, Sarkar Gobinda
Department of Orthopedic Research, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
J Bone Miner Res. 2003 Sep;18(9):1716-22. doi: 10.1359/jbmr.2003.18.9.1716.
Knowledge of the cis-acting elements is required for identifying trans-acting splicing factors underlying cartilage-specific alternative splicing of Col2 pre-mRNA. By performing desired deletions in the mouse Col2 pre-mRNA, location of the intronic cis-acting elements was narrowed down to be at or near splice-junction sequences flanking exon 2 of the gene.
Type II collagen (Col2) pre-mRNA undergoes cartilage-specific alternative splicing involving exon 2 during chondrocyte differentiation. Thus, the trans-acting protein factors that regulate the splicing are associated with the differentiation of chondrocytes. Knowledge of the cognate cis-acting elements is necessary to eventually identify the trans-acting factors.
To localize the cis-acting sequences, we created several deletions within a minigene containing exon 1 to exon 4 of mouse Col 2 gene and evaluated alternative splicing of the resulting pre-mRNAs in ATDC5 cells, a model of insulin-stimulated chondrocyte differentiation. The first deletion reduced intron 1 from 3799 to 259 bp, the second reduced intron 2 from 1108 to 94 bp, the third combined the above two deletions, and the fourth was derived from the third by removing intron 3 and exon 4. ATDC5 cells harboring these constructs were cultured for up to 21 days with or without insulin. Alternative splicing was evaluated by determining the ratio of Col2B (lacks exon 2) to Col2A (has exon 2) RNAs by reverse transcription-polymerase chain reaction.
The deletion in intron 1 had no effect on the alternative splicing while other deletions affected splicing (demonstrated by the presence of splicing intermediates) in cells cultured without insulin or with insulin for 1 week. The splicing intermediates were not seen from any construct when cells were cultured longer (14-21 days) with insulin.
These results show that the 259-bp intron 1, the 94-bp intron 2, and exon 2 sequences retained in the fourth construct provide cis-acting signal sufficient for insulin-induced cartilage-specific alternative splicing of Col2 pre-mRNA.
要确定参与Ⅱ型胶原（Col2）前体mRNA软骨特异性可变剪接的反式作用剪接因子，需要了解顺式作用元件。通过对小鼠Col2前体mRNA进行所需的缺失操作，内含子顺式作用元件的位置被缩小到该基因外显子2侧翼的剪接连接序列处或其附近。
Ⅱ型胶原（Col2）前体mRNA在软骨细胞分化过程中经历涉及外显子2的软骨特异性可变剪接。因此，调节剪接的反式作用蛋白因子与软骨细胞的分化相关。了解相关的顺式作用元件对于最终鉴定反式作用因子是必要的。
为了定位顺式作用序列，我们在一个包含小鼠Col2基因外显子1至外显子4的小基因内创建了几个缺失，并在ATDC5细胞（一种胰岛素刺激的软骨细胞分化模型）中评估所得前体mRNA的可变剪接。第一次缺失将内含子1从3799 bp减少到259 bp，第二次将内含子2从1108 bp减少到94 bp，第三次合并了上述两个缺失，第四次是在第三次的基础上去除内含子3和外显子4得到的。携带这些构建体的ATDC5细胞在有或没有胰岛素的情况下培养长达21天。通过逆转录 - 聚合酶链反应测定Col2B（缺少外显子2）与Col2A（有外显子2）RNA的比例来评估可变剪接。
内含子1的缺失对可变剪接没有影响，而其他缺失在无胰岛素或有胰岛素培养1周的细胞中影响剪接（通过剪接中间体的存在证明）。当细胞用胰岛素培养更长时间（14 - 21天）时，任何构建体中都未观察到剪接中间体。
这些结果表明，第四次构建体中保留的259 bp内含子1、94 bp内含子2和外显子2序列提供了足以实现胰岛素诱导的Col2前体mRNA软骨特异性可变剪接的顺式作用信号。
