Long non-coding RNA Gm15441 attenuates hepatic inflammasome activation in response to metabolic stress

Chad N. Brocker; Donghwan Kim; Tisha Melia; Kritika Karri; Thomas J. Velenosi; Shogo Takahashi; Jessica A. Bonzo; David J. Waxman; Frank J. Gonzalez

Serum and liver triglyceride (TG) levels and liver (but not serum) total cholesterol (CHOL) levels were significantly elevated in fasting Gm15441 LSL mouse livers compared to fasting Gm15441 +/+ mouse livers ( Figure 6C) . These findings were supported by RNA-sequencing analysis, which revealed that liver damage and lipid accumulation-related genes were impacted by Gm15441 deficiency, albeit in a complex manner (Table S2) . Furthermore, analysis of the impact of fasting . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/675785 doi: bioRxiv preprint on mRNAs for apolipoprotein A4 (Apoa4), betaine-homocysteine methyltransferase (Bhmt), lipocalin 2 (Lcn2), orosomucoid 2 (Orm2), serum amyloid A1 (Saa1), and serum amyloid A2 (Saa2) -biomarker genes for liver damage, inflammation, and/or fatty accumulation -showed these genes were significantly elevated in fasting Gm15441 LSL compared to Gm15441 +/+ mouse livers ( Figure 6D ). Fasting induced lower but still significant increases in several of these mRNAs in Gm15441 +/+ livers, which could reflect the fasting-induced increase in Txnip expression ( Figure S2 ). Thus, Gm15441 negatively regulates the NLRP3 inflammasome pathway and lipid accumulation, potentially by preventing Txnip expression, in response to fasting. Txnip translation. To investigate this regulatory mechanism, the translational inhibitory potential of the Txnip 5'UTR was examined when linked to the coding sequence for GFP ( Figure 7A ). The non-5'UTR-GFP and the 5'UTR-GFP expression vectors were each transfected into Hepa-1 cells together with a Gm15441 expression vector or an empty expression plasmid. After 48 hours, strong Gm15441 expression was seen, but had no effect on the RNA levels of GFP or UTR-GFP ( Figure 7B ). In contrast, GFP protein translated from the Txnip 5'UTR-GFP template was significantly reduced in cells co-transfected with Gm15441 expression plasmid ( Figure 7C ). This 5'UTRdependent decrease in GFP protein was most striking when examined by fluorescent microscopy ( Figure 7D and E). These findings suggest Gm15441 may regulate TXNIP translation through IRES sites found within its 5'UTR. To examine whether lncRNA Gm15441 impacts expression of genes that flank Gm15441 on mouse chromosome 3, namely, Hfe2 (Hjv), Pol3gl and Ankrd34a, Hepa-1 cells and NIH3T3 mouse embryonic fibroblast cells were transfected with Gm15441 expression vector, or empty plasmid. The Gm15441 transgene was expressed and significantly downregulated Txnip without impacting the expression of Hfe2, Pol3gl, or Ankrd34a ( Figure 7F ).