title: Rescue of coagulation factor VII function by the U1+5A snRNA.
abstract: Our previous studies with genomic minigenes have demonstrated that an engineered small nuclear RNA-U1 (U1+5a) partially rescued coagulation factor VII (FVII) mRNA processing impaired by the 9726+5G>A mutation. Here, to evaluate the U1+5a effects on FVII function, we devised a full-length FVII splicing-competent construct (pSCFVII-wt). This construct drove in COS-1 cells the synthesis of properly processed FVII transcripts and of secreted functional FVII (23 +/- 4 ng/mL), which were virtually undetectable upon introduction of the 9726+5G>A mutation (pSCFVII-9726+5a). Cotransfection of pSCFVII-9726+5a with pU1+5a resulted in a partial rescue of FVII splicing and protein biosynthesis. The level increase in medium was dose dependent and, with a molar excess (1.5x) of pU1+5a, reached 9.5% plus or minus 3.2% (5.0 +/- 2.8 ng/mL) of FVII-wt coagulant activity. These data provide the first insights into the U1-snRNA-mediated rescue of donor splice sites at protein level, thus further highlighting its therapeutic implications in bleeding disorders, which would benefit even from tiny increase of functional levels.
authors.affiliation: Department of Biochemistry, University of Ferrara, Italy. pnm@unife.it
authors.forename: Mirko
authors.initials: M
authors.lastname: Pinotti
authors.forename: Dario
authors.initials: D
authors.lastname: Balestra
authors.forename: Lara
authors.initials: L
authors.lastname: Rizzotto
authors.forename: Iva
authors.initials: I
authors.lastname: Maestri
authors.forename: Franco
authors.initials: F
authors.lastname: Pagani
authors.forename: Francesco
authors.initials: F
authors.lastname: Bernardi
mesh_terms: D000818:Animals; D019556:COS Cells / Q000378:metabolism; D002522:Chlorocebus aethiops; D005167:Factor VII / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D005813:Genes, Synthetic; D005818:Genetic Engineering; D006801:Humans; D017354:Point Mutation; D012313:RNA / Q000235:genetics / Q000502:physiology; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
chemical_list: D022821:RNA Splice Sites; D012342:RNA, Small Nuclear; C039919:RNA, recombinant; D011993:Recombinant Fusion Proteins; C405483:U1 small nuclear RNA; D012313:RNA; D005167:Factor VII