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Genetic variant in 3' untranslated region of the mouse pycard gene regulates inflammasome activity.

ABSTRACT: Quantitative trait locus mapping for interleukin-1β release after inflammasome priming and activation was performed on bone-marrow-derived macrophages (BMDM) from an AKRxDBA/2 mouse strain intercross. The strongest associated locus mapped very close to the Pycard gene on chromosome 7, which codes for the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). The DBA/2 and AKR Pycard genes only differ at a single-nucleotide polymorphism (SNP) in their 3' untranslated region (UTR). DBA/2 vs. AKR BMDM had increased levels of Pycard mRNA expression and ASC protein, and increased inflammasome speck formation, which was associated with increased Pycard mRNA stability without an increased transcription rate. CRISPR/Cas9 gene editing was performed on DBA/2 embryonic stem cells to change the Pycard 3'UTR SNP from the DBA/2 to the AKR allele. This single base change significantly reduced Pycard expression and inflammasome activity after cells were differentiated into macrophages due to reduced Pycard mRNA stability.

SUBMITTER: Ritchey B

PROVIDER: S-EPMC8248980 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Genetic variant in 3' untranslated region of the mouse pycard gene regulates inflammasome activity.

Ritchey Brian B Hai Qimin Q Han Juying J Barnard John J Smith Jonathan D JD

eLife 20210701

Quantitative trait locus mapping for interleukin-1β release after inflammasome priming and activation was performed on bone-marrow-derived macrophages (BMDM) from an AKRxDBA/2 mouse strain intercross. The strongest associated locus mapped very close to the Pycard gene on chromosome 7, which codes for the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). The DBA/2 and AKR Pycard genes only differ at a single-nucleotide polymorphism (SNP) i ...[more]

PMID: 34197316

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Project description:BackgroundWe previously identified a functional variant in a let-7 microRNA (miRNA) complementary site in the 3'-untranslated region of the KRAS oncogene (rs61764370) which is associated with cancer. We aimed to investigate the association of this KRAS variant with breast cancer and tumour biology.MethodsWe assessed frequency distributions of the KRAS variant in 415 patients with histologically confirmed breast cancer and 457 controls from Connecticut, USA (study group 1) and association of this variant with breast-cancer subtypes in 690 Irish women with known oestrogen receptor (ER), progesterone receptor (PR), and HER2 statuses, and 360 controls (study group 2). We pooled data for study groups 1 and 2 with a cohort of 140 women with triple-negative breast cancer and 113 controls to assess the association of the KRAS variant with triple-negative breast cancer risk, and genome-wide mRNA and specific miRNA expression in patients with triple-negative breast cancer.FindingsAlthough frequency distributions of the KRAS variant in study group 1 did not differ between all genotyped individuals, eight (33%) of 24 premenopausal women with ER/PR-negative cancer had the KRAS variant, compared with 27 (13%) of 201 premenopausal controls (p=0.015). In study group 2, the KRAS variant was significantly enriched in women with triple-negative breast cancer (19 [21%] of 90 cases) compared with 64 (13%) of 478 for luminal A, 13 (15%) of 87 for luminal B, and two (6%) of 35 for HER2-positive subgroups (p=0.044). Multivariate analysis in the pooled study groups showed that the KRAS variant was associated with triple-negative breast cancer in premenopausal women (odds ratio 2.307, 95% CI 1.261-4.219, p=0.0067). Gene-expression analysis of triple-negative breast-cancer tumours suggested that KRAS-variant positive tumours have significantly altered gene expression, and are enriched for the luminal progenitor and BRCA1 deficiency signatures. miRNA analysis suggested reduced levels of let-7 miRNA species in KRAS-variant tumours.InterpretationThe KRAS variant might be a genetic marker for development of triple-negative breast cancer in premenopausal women, and altered gene and miRNA expression signatures should enable molecular and biological stratification of patients with this subgroup of breast cancer.FundingUS National Institutes of Health.

Project description:The endonucleolytic cleavage step of the eukaryotic mRNA 3'-end processing is considered imprecise, which leads to heterogeneity of cleavage site (CS) with hitherto unknown function. Contrary to popular belief, we show that this imprecision in the cleavage is tightly regulated, resulting in the CS heterogeneity (CSH) that controls gene expression in antioxidant response. CSH centres around a primary CS, followed by several subsidiary cleavages determined by CS's positions. Globally and using reporter antioxidant mRNA, we discovered an inverse relationship between the number of CS and the gene expression, with the primary CS exhibiting the highest cleavage efficiency. Strikingly, reducing CSH and increasing primary CS usage induces gene expression. Under oxidative stress (we employ three conditions that induce antioxidant response, tBHQ, H2O2, and NaAsO2) conditions, there is a decrease in the CSH and an increase in the primary CS usage to induce antioxidant gene expression. Key oxidative stress response genes (NQO1, HMOX1, PRDX1, and CAT) also show higher CSH compared to the non-stress response genes and that the number of CSs are reduced to impart cellular response to oxidative stresses. Concomitantly, ectopic expression of one of the key antioxidant response gene (NQO1) driven by the primary CS but not from other subsidiary CSs, or reduction in CSH imparts tolerance to cellular oxidative stresses (H2O2, and NaAsO2). Genome-wide CS analysis of stress response genes also shows a similar result. Compromised CSH or CSH-mediated gene control hampers cellular response to oxidative stress. We establish that oxidative stress induces affinity/strength of cleavage complex assembly, increasing the fidelity of cleavage at the primary CS, thereby reducing CSH inducing antioxidant response. Together, our study reports a novel cleavage imprecision- or CSH-mediated anti-oxidant response mechanism that is distinct and operates downstream but in concert with the transcriptional pathway of oxidative stress induction.

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Genetic variant in 3' untranslated region of the mouse pycard gene regulates inflammasome activity.

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Genetic variant in 3' untranslated region of the mouse <i>pycard</i> gene regulates inflammasome activity.

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