University of Southampton School of Medicine, Division of Human Genetics, Southampton SO16 6YD, UK.
Nucleic Acids Res. 2011 Sep 1;39(16):7077-91. doi: 10.1093/nar/gkr306. Epub 2011 May 23.
GC 5' splice sites (5'ss) are present in ∼1% of human introns, but factors promoting their efficient selection are poorly understood. Here, we describe a case of X-linked agammaglobulinemia resulting from a GC 5'ss activated by a mutation in BTK intron 3. This GC 5'ss was intrinsically weak, yet it was selected in >90% primary transcripts in the presence of a strong and intact natural GT counterpart. We show that efficient selection of this GC 5'ss required a high density of GAA/CAA-containing splicing enhancers in the exonized segment and was promoted by SR proteins 9G8, Tra2β and SC35. The GC 5'ss was efficiently inhibited by splice-switching oligonucleotides targeting either the GC 5'ss itself or the enhancer. Comprehensive analysis of natural GC-AG introns and previously reported pathogenic GC 5'ss showed that their efficient activation was facilitated by higher densities of splicing enhancers and lower densities of silencers than their GT 5'ss equivalents. Removal of the GC-AG introns was promoted to a minor extent by the splice-site strength of adjacent exons and inhibited by flanking Alu repeats, with the first downstream Alus located on average at a longer distance from the GC 5'ss than other transposable elements. These results provide new insights into the splicing code that governs selection of noncanonical splice sites.
GC 5' 剪接位点(5'ss)存在于人类内含子的约 1%中,但促进其有效选择的因素知之甚少。在这里,我们描述了一个由 BTK 内含子 3 中的突变激活的 GC 5'ss 引起的 X 连锁无丙种球蛋白血症的病例。这个 GC 5'ss 本质上很弱,但在存在强而完整的天然 GT 对应物的情况下,它在>90%的初级转录本中被选择。我们表明,这种 GC 5'ss 的有效选择需要在exonized 片段中含有高密 GAA/CAA 剪接增强子,并由 SR 蛋白 9G8、Tra2β 和 SC35 促进。GC 5'ss 可以被靶向 GC 5'ss 本身或增强子的剪接转换寡核苷酸有效抑制。对天然 GC-AG 内含子和以前报道的致病性 GC 5'ss 的综合分析表明,它们的有效激活是通过比其 GT 5'ss 等效物更高密度的剪接增强子和更低密度的沉默子来促进的。相邻外显子的剪接位点强度和侧翼 Alu 重复序列促进了 GC-AG 内含子的去除,但程度较小,而第一个下游 Alu 位于 GC 5'ss 的距离比其他转座元件平均更长。这些结果为指导非规范剪接位点选择的剪接密码提供了新的见解。