Abstract
The human OAS1 (hOAS1) gene produces multiple possible isoforms due to alternative splicing events and sequence variation among individuals, some of which affect splicing. The unique C-terminal sequences of the hOAS1 isoforms could differentially affect synthetase activity, protein stability, protein partner interactions and/or cellular localization. Recombinant p41, p42, p44, p46, p48, p49 and p52 hOAS1 isoform proteins expressed in bacteria were each able to synthesize trimer and higher order 2′-5′ linked oligoadenylates in vitro in response to poly(I:C). The p42, p44, p46, p48 and p52 isoform proteins were each able to induce RNase-mediated rRNA cleavage in response to poly(I:C) when overexpressed in HEK293 cells. The expressed levels of the p42 and p46 isoform proteins were higher than those of the other isoforms, suggesting increased stability in mammalian cells. In a yeast two-hybrid screen, Fibrillin1 (FBN1) was identified as a binding partner for hOAS1 p42 isoform, and Supervillin (SVIL) as a binding partner for the p44 isoform. The p44-SVIL interaction was supported by co-immunoprecipitation data from mammalian cells. The data suggest that the unique C-terminal regions of hOAS1 isoforms may mediate the recruitment of different partners, alternative functional capacities and/or different cellular localization.
Highlights
Oligoadenylate synthetase (OAS) proteins are cellular, double-stranded RNA sensors that function as part of the innate immune response to virus infections [1,2]
Seven isoforms of human OAS1 (hOAS1), p41, p42, p44, p46, p48, p49 and p52, were detected after amplification and cloning of hOAS1 cDNAs from cellular RNA extracted from IFNβ-treated human CCL-110 or CCL-66 fibroblasts
Clear induction of the hOAS1 fusion protein expression could not be detected by Coomassie blue staining following 10% SDS-PAGE of the bacterial extracts, proteins of the expected molecular masses were detected in the induced bacterial lysates by Western blotting with anti-V5 antibody, confirming the expression of these fusion proteins (Figure 2A)
Summary
Oligoadenylate synthetase (OAS) proteins are cellular, double-stranded RNA (dsRNA) sensors that function as part of the innate immune response to virus infections [1,2]. The OAS family consists of one copy each of the OAS1, OAS2, OAS3 and OASL genes. The OAS1 protein contains a single OAS domain, while the OAS2 and OAS3 proteins are composed of two and three. Human OASL contains an inactive OAS domain plus two domains of ubiquitin-like sequences [4,5,6]. Interaction with dsRNA or base-paired regions in a single-stranded. RNA (ssRNA) activates OAS proteins to polymerize ATP into 20 -50 -linked oligoadenylates (2-5A). Activated RNase L cleaves viral and cellular ssRNAs, including 28S and 18S ribosomal
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