Profiling of RNA-binding Proteins Interacting With Glucagon and Adipokinetic Hormone mRNAs

Seungbeom Ko; Eunbyul Yeom; Yoo Lim Chun; Hyejin Mun; Marina Howard-McGuire; Nathan T Millison; Junyang Jung; Kwang-Pyo Lee; Changhan Lee; Kyu-Sun Lee; Joe R Delaney; Je-Hyun Yoon

doi:10.12997/jla.2022.11.1.55

Profiling of RNA-binding Proteins Interacting With Glucagon and Adipokinetic Hormone mRNAs

J Lipid Atheroscler. 2022 Jan;11(1):55-72. doi: 10.12997/jla.2022.11.1.55. Epub 2021 Jul 29.

Authors

Affiliations

¹ Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA.
² Neurophysiology and Metabolism Research Group, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea.
³ Department of Biomedical Science, Graduation School, Kyung Hee University, Seoul, Korea.
⁴ Department of Anatomy and Neurobiology, College of Medicine, Kyung Hee University, Seoul, Korea.
⁵ Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea.
⁶ Leonard Davis School of Gerontology, University of Southern California, Los Angeles, CA, USA.

Abstract

Objective: Glucagon in mammals and its homolog (adipokinetic hormone [AKH] in Drosophila melanogaster) are peptide hormones which regulate lipid metabolism by breaking down triglycerides. Although regulatory mechanisms of glucagon and AKH expression have been widely studied, post-transcriptional gene expression of glucagon has not been investigated thoroughly. In this study, we aimed to profile proteins binding with Gcg messenger RNA (mRNA) in mouse and Akh mRNA in Drosophila.

Methods: Drosophila Schneider 2 (S2) and mouse 3T3-L1 cell lysates were utilized for affinity pull down of Akh and Gcg mRNA respectively using biotinylated anti-sense DNA oligoes against target mRNAs. Mass spectrometry and computational network analysis revealed mRNA-interacting proteins residing in functional proximity.

Results: We observed that 1) 91 proteins interact with Akh mRNA from S2 cell lysates, 2) 34 proteins interact with Gcg mRNA from 3T3-L1 cell lysates. 3) Akh mRNA interactome revealed clusters of ribosomes and known RNA-binding proteins (RBPs). 4) Gcg mRNA interactome revealed mRNA-binding proteins including Plekha7, zinc finger protein, carboxylase, lipase, histone proteins and a cytochrome, Cyp2c44. 5) Levels of Gcg mRNA and its interacting proteins are elevated in skeletal muscles isolated from old mice compared to ones from young mice.

Conclusion: Akh mRNA in S2 cells are under active translation in a complex of RBPs and ribosomes. Gcg mRNA in mouse precursor adipocyte is in a condition distinct from Akh mRNA due to biochemical interactions with a subset of RBPs and histones. We anticipate that our study contributes to investigating regulatory mechanisms of Gcg and Akh mRNA decay, translation, and localization.

Keywords: Glucagon; Mass spectrometry; RNA-binding proteins.

Abstract

Grants and funding