F5-Atlanta induced Exitron Splicing

Introduction:

The factor V (FV)-short-related bleeding disorders, F5-Atlanta (F5-ATL) and east Texas bleeding disorder (ET), provide a striking example of the profound effect of exonic introns, or exitrons, on gene expression, proteomic diversity, and human disease. In F5-ATL and ET, removal of an exitron in exon 13 of the F5 pre-mRNA (F5E13) results in the production of FV-short, a FV protein isoform with high affinity for tissue factor pathway inhibitor α. While the ET missense variant enhances FV-short transcript expression 22-fold, the F5-ATL deletion induces near uniform removal of the F5E13 exitron, driving FV-short production even higher. Presumably, splicing regulatory elements capable of augmenting F5E13 exitron splicing are encoded within F5E13 at the region of F5-ATL. As exitrons have only recently been recognized, few have been functionally characterized and little is known about the molecular determinants governing their retention or removal.

Methods:

To extend our investigations into F5E13 exitron splicing, we developed a novel green fluorescence protein (GFP) based reporter system. In this system, the eukaryotic GFP gene is divided into 2 functional exons, flanked by strong splice sites and intervened by the minute virus of mice (MVM) intron. The F5E13 exitron, defined by the originally annotated ET splice sites, is embedded within the 2nd functional GFP exon. Therefore, the predicted pathway to restoring GFP fluorescence is removal of the F5E13 exitron. Three mini-gene constructs were made: 1 incorporating the wild-type (WT) F5 exitron sequence, 1 incorporating the F5-ATL deletion, and 1 incorporating 3 F5E13 single nucleotide variants that are co-inherited as part of the F5 G-allele haplotype. The mini-gene constructs were stably transfected into FLP-IN HEK293T cells and analyzed for F5E13 splicing by flow cytometry and reverse transcription (RT) standard (s) and quantitative (q) PCR.

Results:

Cells transfected with the F5-ATL reporter demonstrated a significantly higher median fluorescence intensity (MFI) compared to those transfected with WT F5 and F5 G-allele reporters (Welch ANOVA p < 0.0001, Dunnetts T3 p = 0.022 and 0.025). No difference in MFI was seen between the WT F5 and F5 G-allele reporters. Using 2 primer set, RT-sPCR and RT-qPCR were used to analyze differential transcript expression among the variants. Using primers flanking the F513 exitron, RT-sPCR detected prominent spliced transcripts in all of the reporter variants. Un-spliced F5E13 transcripts were detected in cells expressing WT F5 and F5 G-allele, but not F5-ATL, reporters. Furthermore, F5-ATL was associated with significantly higher total reporter transcripts than WT F5 or F5 G-allele (Welch ANOVA p = 0.007, Dunnetts T3 p = 0.017 and 0.036). Specifically, the F5-ATL reporter generated 20-fold more total transcripts than the WT F5 reporter. No significant difference was observed between the WT F5 and F5 G-allele constructs. Intriguingly, F5-ATL reporter transcript levels also trended towards being higher than the GFP control reporter, suggesting a positive overall effect of F5-ATL exitron splicing on gene expression independent of the F5 gene locus.

Discussion/Conclusions

Exitrons are emerging as important contributors to gene expression and proteomic diversity. However, few well-characterized human examples exist and of these, the majority are associated with cancer progression. Therefore, the F5E13 exitron represents a unique opportunity to study basic exitron biology as well as the pathway to FV-short and hemostatic balance. Using a reductionist reporter model, our results demonstrate the ability of the F5E13 exitron to undergo splicing in a synthetic system. Furthermore, as observed with the original F5-ATL proband, F5-ATL confers near-uniform exitron removal in our reporter model. Surprisingly, inclusion of F5-ATL within the F5E13 exitron supported total transcript levels trending above that of the positive exitron-less GFP control. This finding correlates with the marked overexpression of FV-short previously described in the F5-ATL proband and suggests a potential role for F5-ATL and F5E13 exitron splicing in overall gene expression regulation. Remaining questions to be addressed include the i) role of F5E13 exitron in normal hemostasis, ii) identification of the regulatory mechanisms governing F5E13 exitron splicing, and iii) the evolutionary history of the F5E13 exitron.

Zimowski:Hema Biologics: Consultancy; CSL Behring: Consultancy; Pfizer: Research Funding; CSL Behring: Research Funding; Hemophilia of Georgia: Honoraria, Research Funding; Octapharma: Other: Travel Funds. Meeks:Regeneron: Consultancy; Pfizer: Consultancy; Genentech: Research Funding; Biomarin: Consultancy; Octapharma: Research Funding; Sanofi: Consultancy; Bayer: Consultancy; Genentech: Consultancy. Doering:Expression Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.