Finely tuned differential expression of alternative splice variants contributes to important physiological processes such as the fine-tuning of electrical firing or hearing frequencies; yet the underlying molecular basis for the expression control is not clear. The inclusion levels of four depolarization-regulated alternative exons were measured by RT-PCR in GH3 pituitary cells under different conditions of stimulation and/or RNA interference of splicing factors. The usage of the exons was reduced by membrane depolarization to various extents and was differentially modulated by the knock-down of splicing factors hnRNP L, L-like, I (PTBP1) or K or their combinations. A spectrum of each exon's level was produced under six knock-down conditions and was significantly shifted by depolarization. When all these conditions were considered together, a more refined or expanded spectrum of exon usage was obtained for each of the four exons. As a proof of principle for the molecular basis of the fine-tuning of exon usage, we show in the cases of hnRNP L and LL that their differential effects through the same element or different combinations of RNA sequences by the same factor hnRNP L are critical. The results thus demonstrate that the combined effect of varying extracellular stimuli and intracellular factors/RNA sequences refines or expands the spectra of endogenous exon usage, likely contributing to the fine-tuning of cellular properties.
Keywords: Alternative splicing; Membrane depolarization; PTBP1; hnRNP K; hnRNP L/LL.
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