Submitted March 3, 2009
Accepted April 16, 2009
Rescue of coagulation factor VII function by the U1+5A snRNA
Mirko Pinotti*, Dario Balestra, Lara Rizzotto, Iva Maestri, Franco Pagani, and Francesco Bernardi
Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy
Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy
* Corresponding author; email: pnm{at}unife.it.
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±4ng/ml), which were virtually undetectable upon introduction of the 9726+5g/a mutation (pSCFVII-9726+5a).
Co-transfection 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±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.
