The Differential Translation Capabilities of the Human DHFR2 Gene Indicates a Developmental and Tissue-Specific Endogenous Protein of Low Abundance

Identifier:  imarina:9390010

Handle: https://hdl.handle.net/20.500.11797/imarina9390010

Authors:  Bookey, N; Drago, P; Leung, KY; Hughes, L; MacCooey, A; Ozaki, M; Henry, M; De Castro, SCP; Doykov, I; Heywood, WE; Mills, K; Murphy, MM; Cavalle-Busquets, P; Campbell, S; Burtenshaw, D; Meleady, P; Cahill, PA; Greene, NDE; Parle-McDermott, A

Abstract:
A functional role has been ascribed to the human dihydrofolate reductase 2 ( DHFR2 ) gene based on the enzymatic activity of recombinant versions of the predicted translated protein. However, the in vivo function is still unclear. The high amino acid sequence identity (92%) between DHFR2 and its parental homolog, DHFR, makes analysis of the endogenous protein challenging. This paper describes a targeted mass spectrometry proteomics approach in several human cell lines and tissue types to identify DHFR2-specific peptides as evidence of its translation. We show definitive evidence that the DHFR2 activity in the mitochondria is in fact mediated by DHFR, and not DHFR2. Analysis of Ribo-seq data and an experimental assessment of ribosome association using a sucrose cushion showed that the two main Ensembl annotated mRNA isoforms of DHFR2, 201 and 202, are differentially associated with the ribosome. This indicates a functional role at both the RNA and protein level. However, we were unable to detect DHFR2 protein at a detectable level in most cell types examined despite various RNA isoforms of DHFR2 being relatively abundant. We did detect a DHFR2-specific peptide in embryonic heart, indicating that the protein may have a specific role during embryogenesis. We propose that the main functionality of the DHFR2 gene in adult cells is likely to arise at the RNA level.