The dihydrofolate reductase gene family consists of two functional genes. The DHFR gene is the more researched gene of this family. It encodes an enzyme that reduces dihydrofolate to tetrahydrofolate in a cell. This reduction is an important step in the onecarbon metabolism pathway, which is responsible for purine/thymidylate biosynthesis and homocysteine re-methylation. The remaining functional gene is known as the DHFR2 gene. There was very little knowledge about this retrogene prior to this project. Given the importance of the DHFR gene, it was vital the function of the DHFR2 gene was elucidated. Therefore this thesis aimed to characterise it by focusing on the expression and behaviours of its RNA and protein. Firstly, the translatability of the DHFR2 transcripts was assessed by performing a sucrose cushion ultracentrifugation RT-qPCR experiment. The two main DHFR2 transcripts showed differential translational capabilities, with one of them potentially encoding a protein. Secondly, the expression of the endogenous DHFR2 protein was investigated using LC-MS/MS. There was no evidence of the native DHFR2 protein in the human cells/tissues examined, apart from heart tissue from early development. This result suggests that the DHFR2 gene encodes a protein to supply dihydrofolate reductase activity to rapidly growing embryos and foetuses. Thirdly, mechanisms of translational regulation were considered for the DHFR2 transcripts as the native protein had not been detected in all the cells/tissues examined. There was evidence that some of the DHFR2 transcripts were being translationally inhibited via paraspeckle nuclear retention. Finally, an additional role for the DHFR2 gene was investigated as the DHFR2 RNA was found to be highly abundant in multiple cells/tissues despite the protein being undetectable in the same samples. Mass spectrometry-based differential analysis on a DHFR2 knockout and overexpression model identified that the DHFR2 RNA has a role in regulating the expression of the DHFR gene. To conclude, the results gathered in this thesis revealed the biological roles of the human DHFR2 gene. These significant findings will pave the way for future research on this functional gene which may have a major impact on human health and disease.