U1-snRNA-mediated rescue of mRNA processing in severe factor VII deficiency.
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.
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D005168:Factor VII Deficiency / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics*
D012333:RNA, Messenger; D012342:RNA, Small Nuclear; C405483:U1 small nuclear RNA