Poster abstracts
Poster number 183 submitted by Anchal Agarwal
Transcriptome and eCLIP-seq analyses of eIF2A targets in 3T3-L1 mouse cells
Anchal Agarwal (Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, OH), Richard Anderson, Archana Prabahar, Barsanjit Mazumder, Peng Jiang (Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, OH), Frank Tedeschi, Soon Yeul Yi, (Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH), Bo-Jhih Guan, William C. Merrick, David A. Buchner, Maria Hatzoglou (Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH ), Anton A. Komar (Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, OH; Department of Biochemistry, Case Western Reserve University, Cleveland, OH )
Abstract:
We have previously provided evidence implicating eukaryotic initiation factor 2A (eIF2A) in the control of lipid metabolism in eIF2A-KO mice. We, in particular, found that primary adipocytes isolated from eIF2A-KO mice reveal increased lipid accumulation upon differentiation in comparison with primary adipocytes isolated from wild-type mice. To gain further insights into the role of eIF2A in lipid metabolism, we used the embryonic fibroblast cell line 3T3-L1, which is commonly utilized for the analysis of the subcellular pathways involved in preadipocyte cell differentiation and lipogenesis. Interestingly, we found that eIF2A depletion in 3T3-L1 cells results in a more than 2-fold decrease in lipid accumulation upon cellular differentiation from preadipocytes to adipocytes, opposite to that observed in mice, where an increase in lipid accumulation has been observed. RNA-seq Gene Ontology and KEGG enrichment analyses revealed that the decrease in lipid accumulation in eIF2A-depleted cells is not related to the differences in the adipogenic differentiation program per se but rather is associated with the changes in expression of selected mRNA transcripts in insulin and glucagon signaling pathways as well as pathways controlling fatty acid metabolism. The difference between in vivo and ex vivo experiments suggests that external stimuli in vivo in mice and ex vivo might affect adipocytes differently. Nevertheless, both results clearly indicate that eIF2A is involved in the control of lipid metabolism. eCLIP-seq analysis revealed many unexpected eIF2A RNA targets such as miRNAs, ncRNAs, lincRNAs, and SnoRNAs, besides mRNAs. Surprisingly, the binding of eIF2A to mRNAs was not limited to the 5UTRs (as would be expected, given eIF2A's role as an initiation factor bound to 40S ribosomal subunits), but was also found to occur in the open reading frames and, even more surprisingly, in the 3UTRs of the target mRNAs. Among the many genes affected, Lars2 (leucyl-tRNA synthetase 2, mitochondrial) has 99.9% eIF2A binding in its 3UTR. The reporter assays involving a fusion of the Lars2 3UTR to luciferase uncovered a novel mechanism by which eIF2A may affect gene expression programs.
References:
Anderson, R., Agarwal, A., et.al (2021). eIF2A-knockout mice reveal decreased life span and metabolic syndrome. FASEB J, 2021.
Keywords: eIF2A, lipid metabolism, eCLIP-seq