Raponi Michela, Buratti Emanuele, Dassie Elisa, Upadhyaya Meena, Baralle Diana
Department of Pathology, University of Cambridge, UK.
FEBS J. 2009 Apr;276(7):2060-73. doi: 10.1111/j.1742-4658.2009.06941.x.
Many disease-causing splicing mutations described in the literature produce changes in splice sites (SS) or in exon-regulatory sequences. The delineation of these splice aberrations can provide important insights into novel regulation mechanisms. In this study, we evaluated the effect of patient variations in neurofibromatosis type 1 (NF1) exon 29 and its 5'SS surrounding area on its splicing process. Only two of all nonsense, missense, synonymous and intronic variations analyzed in this study clearly altered exon 29 inclusion/exclusion levels. In particular, the intronic mutation +5g>a had the strongest effect, resulting in total exon exclusion. This finding prompted us to evaluate the exon 29 5'SS in relation to its ability to bind U1 snRNP. This was performed by direct analysis of the ability of U1 to bind to wild-type and mutant donor sites, by engineering an in vitro splicing system to directly evaluate the functional importance of U1 snRNA base pairing with the exon 29 donor site, and by coexpression of mutant U1 snRNP molecules to try to rescue exon 29 inclusion in vivo. The results revealed a low dependency on the presence of U1 snRNP, and suggest that exon 29 donor site definition may depend on alternative mechanisms of 5'SS recognition.
文献中描述的许多致病剪接突变会导致剪接位点（SS）或外显子调控序列发生变化。这些剪接异常的描绘可为新型调控机制提供重要见解。在本研究中，我们评估了1型神经纤维瘤病（NF1）外显子29及其5'剪接位点周围区域的患者变异对其剪接过程的影响。在本研究分析的所有无义、错义、同义及内含子变异中，只有两个明显改变了外显子29的包含/排除水平。特别是，内含子突变+5g>a的影响最强，导致外显子完全排除。这一发现促使我们评估外显子29的5'剪接位点与结合U1 snRNP能力的关系。这是通过直接分析U1与野生型和突变型供体位点结合的能力、构建体外剪接系统以直接评估U1 snRNA与外显子29供体位点碱基配对的功能重要性以及共表达突变型U1 snRNP分子以试图在体内挽救外显子29的包含来进行的。结果显示对U1 snRNP存在的依赖性较低，并表明外显子29供体位点的定义可能取决于5'剪接位点识别的替代机制。
