Murray Patricia, Clegg Roger A, Rees Huw H, Fisher Michael J
School of Biological Sciences, Biosciences Building, University of Liverpool, Liverpool, UK.
Gene. 2008 Jan 31;408(1-2):157-63. doi: 10.1016/j.gene.2007.10.034. Epub 2007 Nov 17.
In C. elegans, the PK-A catalytic subunit is encoded by kin-1, which has six 5' exons (N'1-N'6), any one of which may be alternatively spliced onto exon-2. Here we describe a novel siRNA-based strategy to knockdown the expression levels of the N'3 and N'4 splice variants. We show that this technique can effectively knockdown expression of the targeted isoforms without affecting expression of the other kin-1 splice variants. We suggest that this strategy could be widely used in C. elegans to investigate the function of genes with alternative first exons. Moreover, we report a novel role for the N'3 kin-1 variant. Whereas knockdown of the N'4 variant results in no obvious phenotype, loss of the N'3 variant leads to paralysis and an egg-laying defect in the adult, suggesting a deficit in the function of the neuromuscular junction. The function of the N'3 variant is discussed in relation to the known function of PK-A in regulation of the release of neurotransmitters from many presynaptic termini.
在秀丽隐杆线虫中，PK - A催化亚基由kin - 1编码，kin - 1有六个5'外显子（N'1 - N'6），其中任何一个都可以选择性地剪接到外显子2上。在这里，我们描述了一种基于小干扰RNA（siRNA）的新策略，用于降低N'3和N'4剪接变体的表达水平。我们表明，该技术可以有效降低靶向异构体的表达，而不影响其他kin - 1剪接变体的表达。我们认为，这种策略可广泛应用于秀丽隐杆线虫，以研究具有可变首个外显子的基因的功能。此外，我们报道了N'3 kin - 1变体的一个新作用。虽然敲低N'4变体不会导致明显的表型，但N'3变体的缺失会导致成虫麻痹和产卵缺陷，这表明神经肌肉接头功能存在缺陷。我们结合PK - A在调节许多突触前末端神经递质释放方面的已知功能，讨论了N'3变体的功能。
