Pinotti Mirko, Rizzotto Lara, Balestra Dario, Lewandowska Marzena Anna, Cavallari Nicola, Marchetti Giovanna, Bernardi Francesco, Pagani Franco
Department of Biochemistry and Molecular Biology, University of Ferrara, Via Fossato di Mortara 74, Ferrara, Italy.
Blood. 2008 Mar 1;111(5):2681-4. doi: 10.1182/blood-2007-10-117440. Epub 2007 Dec 21.
Small nuclear U1-RNAs (snRNAs), the spliceosome components selectively recognizing donor splice sites (5'ss), were engineered to restore correct mRNA processing in a cellular model of severe coagulation factor VII (FVII) deficiency, caused by the IVS7 9726 + 5g/a change. Three U1-snRNAs, complementary to the mutated 5'ss (U1 + 5a) or to neighboring sequences were expressed with FVII minigenes in a hepatoma cell line. The U1-snRNAs reduced from 80% to 40% the exon 7 skipping, thus increasing exon definition. The U1 + 5a construct also dramatically increased recognition of the correct 5'ss over the 37-bp downstream cryptic site preferentially activated by the mutation, thus inducing appreciable synthesis of normal transcripts (from barely detectable to 50%). This effect, which was dose-dependent, clearly demonstrated that impaired recognition by the U1-snRNA was the mechanism responsible for FVII deficiency. These findings suggest compensatory U1-snRNAs as therapeutic tools in coagulation factor deficiencies caused by mutations at 5'ss, a frequent cause of severe defects.
小核U1-RNAs（snRNAs）是剪接体的组成部分，可选择性识别供体剪接位点（5'ss）。通过基因工程改造，使其在严重凝血因子VII（FVII）缺乏的细胞模型中恢复正确的mRNA加工过程，该模型由IVS7 9726 + 5g/a突变引起。三种与突变的5'ss（U1 + 5a）或相邻序列互补的U1-snRNAs与FVII小基因在肝癌细胞系中共同表达。U1-snRNAs使外显子7跳跃从80%降至40%，从而增强了外显子的界定。U1 + 5a构建体还显著增加了对正确5'ss的识别，超过了由突变优先激活的37bp下游隐蔽位点，从而诱导了正常转录本的可观合成（从几乎检测不到增加到50%）。这种剂量依赖性效应清楚地表明，U1-snRNA识别受损是FVII缺乏的原因。这些发现提示，补偿性U1-snRNAs可作为治疗由5'ss突变引起的凝血因子缺乏的治疗工具，5'ss突变是严重缺陷的常见原因。
