Jared Lambert and Dr. Brian Poole, Department of Microbiology and Molecular Biology

With our lab’s previous finding revealing a hairpin folding structure in exon 1B (IRF5 risk allele), not found in the other first exons, this project was focused towards more fully understanding the role of IRF5 exon 1B’s hairpin structure and its effect on the translation of downstream proteins. The data acquired through this project will provide much needed information regarding the mechanism through which the risk allele of IRF5 affects the development of Systemic Lupus Erythmatosus (SLE).

Mutational analysis showed that the hairpin shape decreased translation five-fold in a luciferase reporter assay

Using pGL3-Control luciferase plasmid different 5′ UTRs were introduced directly upstream of the luciferase coding region as a substitute 5’ UTR, though all were under the control of the same promoter to have equal levels of transcription, but reveal any differences in translation. HEK cells, which do not express IRF5 normally, were transfected with one of the three luciferase plasmids as well as a DsRed expressing plasmid, which was included as a transfection control. Values for luciferase expression were then normalized to the transfection efficiency. It was found that the hairpin shape caused a 5-fold decrease in translation, when compared to the sequence which was not predicted to form a hairpin. The two sequences which were predicted to form a hairpin structure were in fact translated 5-fold less than the non-hairpin mutant.

Transcripts initiating with exon 1B are not well translated compared to the other first exons of IRF5

Because the hairpin structure inhibited translation, we hypothesized that transcripts containing exon 1B would be translated less efficiently than those beginning with the other first exon choices. Each of the IRF5 first exon variants were inserted into luciferase vectors and used to compare the effect on translation efficiency. An equal level of transcription was assumed as each plasmid had the same promoter. The entire 5’UTR was added in front of the luciferase with sequences exactly matching published IRF5 5’UTRs. Exon 1B was the least well translated, especially when compared to exon 1A (28-fold lower, p=0.048) and when compared to 1D (16-fold lower, p=0.022).

IRF5 identities are varied and differ by risk status

In an effort to visualize how the presence or absence of exon 1B may affect the splicing of the other eight exons, experiments were done to determine identity by sample size analysis, with different sizes representing truncations or altered splicing events. This was done by PCR amplification of the IRF5 variants using a common reverse primer paired to forward primers that were specific for each of the 4 known exons 1. Before PCR amplification, equimolar ratios cDNA from each cell line were pooled into a common risk and a common protective sample. This pooling of the samples facilitated a simple overview of the splicing events that are occurring by size comparison. The size of smaller variants indicate that there are large truncations in the mRNA. This analysis shows that at least in LCLs, IRF5 is spliced differently in risk cells and that the identity as well as the quantity of splice variants differs in risk and protective cells (Fig. 1).

Discussion

The different roles of IRF5 align with the range of autoimmune diseases it is involved in. For instance, IRF5 functions as a cytokine transcription factor and as p53-independent pro-apoptotic regulator. Dysregulation of cytokine levels and apoptosis are features of SLE, the disease which the rs2004640 was first associated. IRF5 has also been shown to be an important mediator in macrophage differentiation towards pro-inflammatory M1 macrophages. With high levels of IRF5 there would be more pro-inflammatory cytokines, which would provoke a greater immune response.

The etiologies of autoimmune diseases are complex, but various genetic and environmental factors contribute to their onset. Studies on genetic risk factors such as the rs2004640 in IRF5 point to the pathways involved in disease and therefore to therapies which will allow for more effective treatment.