Fig 7. Functional characterizations of OAS1 isoforms.

(A) Protein expression of OAS1 isoforms was evaluated in EBV-transformed B cells from SS patients (four independent samples from each genotype group) using anti-OAS1 antibody targeting the shared epitope of all the isoforms. The stimulated cells were treated with universal type I IFN (1500U/ml) for 24hrs. The p44 isoform was not detectable using western-blot due to its low expression. The right panel shows quantified band intensity normalized to the GAPDH in each sample. (B) The transcript levels of each OAS1 isoform from the same sets of cells described above were determined using real-time PCR. Consistent with the RNA-seq results, the SS-associated risk allele A of rs10774671 was correlated with decreased levels of p46 and increased expression of the p42, p48, and p44 isoforms (significance levels are shown at the bottom). The transcript levels of all the isoforms significantly increased after IFN stimulation (two-tailed t test); however, only p46 had increased protein production after IFN stimulation. (Significance level: ** P<0.01; *** P<0.001) (C) Individual isoforms of OAS1 tagged with Xpress epitope were cloned and transfected into HEK 293T cells for 48hrs. The p48 and p44 isoforms had impaired protein expression compared to p46 and p42, although their transcript levels were equivalent as determined by real-time PCR (n = 4; normalized to HMBS). (D) The full-length and truncated OAS1 p48 and p44 isoforms were cloned into HEK 293T cells. Western-blot indicated the lack of expression of the full-length p48 and p44 isoforms, whereas the truncation of both isoform transcripts (T2 and T4) was able to restore protein expression. (E) The 3' alternatively spliced terminus of different OAS1 isoforms were linked to the 3'-end of GFP to observe their influence on GFP protein expression in HEK 293T cells. The 3'-terminus from the p48 and p44 isoforms resulted in decreased expression of GFP.