Silencing of the tRNA Modification Enzyme Cdkal1 Effects Functional Insulin Synthesis in NIT-1 Cells: tRNALys3 Lacking ms2- (ms2t6A37) is Unable to Establish Sufficient Anticodon:Codon Interactions to Decode the Wobble Codon AAG

Amithi Narendran; Sweta Vangaveti; Srivathsan V Ranganathan; Emily Eruysal; Miranda Craft; Omar Alrifai; Fu Yee Chua; Kathryn Sarachan; Breann Litwa; Sheetal Ramachandran; Paul F Agris

doi:10.3389/fmolb.2020.584228

Silencing of the tRNA Modification Enzyme Cdkal1 Effects Functional Insulin Synthesis in NIT-1 Cells: tRNA^Lys3 Lacking ms²- (ms²t⁶A₃₇) is Unable to Establish Sufficient Anticodon:Codon Interactions to Decode the Wobble Codon AAG

Front Mol Biosci. 2021 Feb 9:7:584228. doi: 10.3389/fmolb.2020.584228. eCollection 2020.

Authors

Affiliations

¹ The RNA Institute and Department of Biological Sciences, University of Albany, Albany, NY, United States.
² Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA, United States.
³ Knight Cancer Institute, Oregon Health Sciences, School of Medicine, Portland, OR, United States.
⁴ Department of Medicine, Duke University School of Medicine, Durham, NC, United States.

Abstract

Human Genome Wide Association Studies found a significant risk of Type 2 Diabetes Mellitus (T2DM) in single nucleotide polymorphisms in the cdkal1 gene. The cdkal1 gene is remote from the insulin gene and with the surprising function of a specific tRNA modification. Population studies and case control studies acquired evidences of the connection between Cdkal1 protein and insulin production over the years. To obtain biochemical proofs directly linking potential SNPs to their roles in insulin production and availability is challenging, but the development of Cdkal1 knock out mice and knock out cell lines made it possible to extend our knowledge towards therapeutic field of diabetic research. Supporting the evidences, here we show that knock down of the cdkal1 gene using small interfering and short hairpin RNA in the NIT-1 cell line, a β-cell line inducible for insulin resulted in reduced levels of cdkal1 and mature insulin mRNAs, increased the level of precursor insulin mRNA, decreased Cdkal1 and insulin proteins, and diminished modification of tRNA^Lys3 from t⁶A₃₇ to ms²t⁶A₃₇, the specified function of Cdkal1. tRNA^Lys3 lacking ms²- is incapable of establishing sufficient hydrogen bonding energy and hydrophobic stabilization to decode the wobble codon AAG.

Keywords: CDKAL1; Cdkal1 protein; Type 2 diabetes; Wobble decoding on the ribosome; insulin; tRNA modification.