Pessa Heli K J, Ruokolainen Annukka, Frilander Mikko J
Institute of Biotechnology, Program on Developmental Biology, PL56 (Viikinkaari 9), 00014 University of Helsinki, Finland.
RNA. 2006 Oct;12(10):1883-92. doi: 10.1261/rna.213906. Epub 2006 Sep 6.
The rate of excision of U12-type introns has been reported to be slower than that of U2-type introns, suggesting a rate-limiting bottleneck that could down-regulate genes containing U12-type introns. The mechanistic reasons for this slower rate of intron excision are not known, but lower abundance of the U12-type snRNPs and slower rate of assembly or catalytic activity have been suggested. To investigate snRNP abundance we concentrated on the U4atac snRNA, which is the least abundant of the U12-type snRNAs and is limiting the formation of U4atac/U6atac complex. We identified mouse NIH-3T3 cell line isolates in which the level of both U4atac snRNA and U4atac/U6atac complexes is reduced to 10%-20% of the normal level. We used these cell lines to investigate splicing efficiency by transient transfection of a reporter gene containing a U12-type intron and by quantitative PCR analysis of endogenous genes. The splicing of the reporter U12-type intron was very inefficient, but the activity could be restored by overexpression of U4atac snRNA. Using these U4atac-deficient NIH-3T3 cells, we confirmed the results of previous studies showing that U12-type introns of endogenous genes are, indeed, excised more slowly than U2-type introns, but we found that the rate did not differ from that measured in cells displaying normal levels of U4atac snRNA. Thus our results suggest that the cellular abundance of the snRNPs does not limit U12-type intron splicing under normal conditions.
据报道，U12型内含子的切除速率比U2型内含子慢，这表明存在一个限速瓶颈，可能会下调含有U12型内含子的基因。内含子切除速率较慢的机制原因尚不清楚，但有人提出可能是U12型小核核糖核蛋白（snRNP）丰度较低，以及组装或催化活性速率较慢。为了研究snRNP的丰度，我们重点关注U4atac snRNA，它是U12型snRNA中丰度最低的，并且限制了U4atac/U6atac复合体的形成。我们鉴定出小鼠NIH-3T3细胞系分离株，其中U4atac snRNA和U4atac/U6atac复合体的水平均降至正常水平的10%-20%。我们使用这些细胞系，通过瞬时转染含有U12型内含子的报告基因以及对内源基因进行定量PCR分析，来研究剪接效率。报告基因中U12型内含子的剪接效率非常低，但通过过表达U4atac snRNA可以恢复其活性。使用这些缺乏U4atac的NIH-3T3细胞，我们证实了先前研究的结果，即内源基因的U12型内含子确实比U2型内含子切除得更慢，但我们发现该速率与在U4atac snRNA水平正常的细胞中测得的速率没有差异。因此，我们的结果表明，在正常条件下，snRNP的细胞丰度并不限制U12型内含子的剪接。
