Abstract 3017: Identification and functional characterization of novel splicing variants of the immunoproteasome catalytic subunits B1i and B2i.
Authors: Ao Lin , Park Jieun , Kim Kyungbo , Lee Wooin 
Published: August 2014

Abstract
Background: The immunoproteasome is an alternative form to the standard proteasome containing a distinct set of catalytic subunits, β1i, β2i and β5i. Beyond its well-known role in antigen presentation, the immunoproteasome has been recognized to have important functions in the pathogenesis of cancer and other inflammatory diseases. Alternative splicing is a crucial mechanism for generating protein diversity and functional changes under stress conditions. While it was reported that cancer cells express a nonfunctional splicing variant of β5i, splicing variants of β1i or β2i have not yet been described.
Methods: Using RT-PCR and direct sequencing, we examined whether cancer cells express splicing variants of β1i or β2i. Qualitative and quantitative RT-PCRs were performed using specific primers designed to amplify the wildtype or the variant forms identified. To examine their biological significance, we investigated whether β1i or β2i variants can be incorporated into the assembled proteasome complex. Specifically, we performed co-immunoprecipitation assays using antibodies against other components of the assembled proteasome, α2 and POMP (proteasome maturation protein).
Results: RNA analyses of multiple cancer cell lines detected the presence of β1i and β2i variants, arising from exon skipping or intron retention. The β1i variant lacks exon 3, leading to the deletion of an internal 44 amino acids. The β2i variant is truncated at the C-terminus, as a result of intronic retention between exons 6 and 7. RT-PCR results showed that β1i and β2i variants are expressed at varying levels in cell lines from multiple types of cancer, as well as clinical tumor samples (e.g. colon, pancreatic cancer). Furthermore, quantitative RT-PCR results indicate that the expression of variants can be upregulated by interferon- γ, a known inducer of the immunoproteasome. Using cell lysates transfected with expression plasmids containing the variant coding sequences fused with a myc/FLAG tag, co-immunoprecipitation results suggested that the variant forms of β1i and β2i can be incorporated into the assembled proteasome complex.
Conclusion: We report the identification of two novel splicing variants of the immunoproteasome subunits β1i and β2i. Our results indicate that β1i and β2i variants are expressed in cancer cells and can be incorporated into the assembled proteasome. These findings suggest that β1i and β2i variants may consequently have an impact on the overall catalytic function of the immunoproteasome. Further investigations are ongoing to assess the proteolytic activity of immunoproteasomes containing β1i and β2i variants using subunit-selective probe substrates. Taken together, our findings may provide further insights into mechanisms by which cancer cells regulate the expression and function of the immunoproteasome.
Citation Format: Lin Ao, Jieun Park, Kyungbo Kim, Wooin Lee. Identification and functional characterization of novel splicing variants of the immunoproteasome catalytic subunits B1i and B2i. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3017. doi:10.1158/1538-7445.AM2013-3017