Correct mRNA Processing at a Mutant TT Splice Donor in FANCC Ameliorates the Clinical Phenotype in Patients and Is Enhanced by Delivery of Suppressor U1 snRNAs

Abstract

The U1 small nuclear RNA (U1 snRNA) as a component of the major U2-dependent spliceosome recognizes 5' splice sites (5'ss) containing GT as the canonical dinucleotide in the intronic positions +1 and +2. The c.165+1G>T germline mutation in the 5'ss of exon 2 of the Fanconi anemia C (FANCC) gene commonly predicted to prevent correct splicing was identified in nine FA patients from three pedigrees. RT-PCR analysis of the endogenous FANCC mRNA splicing pattern of patient-derived fibroblasts revealed aberrant mRNA processing, but surprisingly also correct splicing at the TT dinucleotide, albeit with lower efficiency. This consequently resulted in low levels of correctly spliced transcript and minute levels of normal posttranslationally processed FANCD2 protein, indicating that this naturally occurring TT splicing might contribute to the milder clinical manifestations of the disease in these patients. Functional analysis of this FANCC 5'ss within splicing reporters revealed that both the noncanonical TT dinucleotide and the genomic context of FANCC were required for the residual correct splicing at this mutant 5'ss. Finally, use of lentiviral vectors as a delivery system to introduce expression cassettes for TT-adapted U1 snRNAs into primary FANCC patient fibroblasts allowed the correction of the DNA-damage-induced G2 cell-cycle arrest in these cells, thus representing an alternative transcript-targeting approach for genetic therapy of inherited splice-site mutations.

Widespread recognition of 5′ splice sites by noncanonical base-pairing to U1 snRNA involving bulged nucleotides

“…S1A). This is in contrast to what was reported for a similar mutant 59ss in FANCC (AUG/UUAAGUAG, where ''/'' is the mapped exon/intron boundary) (Hartmann et al 2010), and consistent with a mutation that creates a 59ss in SMARCAD1 (ACU/GUUAAGUAC), associated with autosomal-dominant adermatoglyphia (lack of fingerprints) (Nousbeck et al 2011).…”

“…We determined the identity of each PCR product by subcloning agarose gel-purified bands with an Original TA Cloning kit (Invitrogen) followed by sequencing. We also directly sequenced the RT-PCR products in Figure 1D to test for potential splicing at a UU 59ss dinucleotide, as reported for 59ss similar to the atypical 59ss (Hartmann et al 2010). …”

Fanconi anemia: a model disease for studies on human genetics and advanced therapeutics

“…FA-IPS cells offer a novel tool to model FA physiology and pathogenesis and provide a cell platform for drug screening [96]. Finally, therapies designed to enhance the correct mRNA processing at a mutant TT splice donor in FANCC using suppressor U1 snRNAs, suggests that correcting pathological mRNA processing at specific mutant splice sites might apply to FA complementation groups in a mutation-specific fashion [103]. Therefore, our better understanding of the molecular genetic defects of FA and the use of gene correction strategies may contribute to futures treatments for this devastating disease.…”

mRNA Transcript Diversity Creates New Opportunities for Pharmacological Intervention

“…Likewise, spliceosome-adapted U7 snRNA is needed to process histone mRNA. Modified synthetic U1 and U7 RNAs were shown to be effective in cell culture at inducing exon skipping or inclusion (Pinotti et al, 2008;Geib and Hertel, 2009;Hartmann et al, 2010;Incitti et al, 2010). A means of targeting specific mRNA transcript isoforms relies on complementary antisense oligonucleotides that can restore the normal splicing process disrupted by a mutation, skip disease-associated exons (Skordis et al, 2003), degrade unwanted transcripts, or increase production of a desired transcript.…”