Discussion

Synonymous codons are not used with the same frequencies within and between different species, generating codon bias. However, the extent of which codon preference impacts on gene expression in trypanosomatids is unclear. In this work we developed a novel codon usage metric called the 'gene expression codon adaptation index' (geCAI) that allows a prediction of mRNA abundance with a coefficient of determination of 0.55. The geCAI metric was validated using a synthetic library of 22 synonymous GFP sequences in procyclic T. brucei. Protein expression and mRNA levels were modulated over a ~40-fold range, similar to range measured for endogenous genes. Altering the geCAI value increased the expression level of a VSG mRNA. Investigation of mRNA turnover showed that GFPs with low geCAI values had shorter half-lives, indicating that translational efficiency impacts on mRNA steady-state levels. Impairment of translation led to stabilisation of GFP mRNAs whereas reducing translation initiation frequency had the opposite effect, corroborating the role of translation in regulation of mRNA levels. Analysis of the effect of codon position showed that introduction of rare codons reduced both protein and mRNA levels independently of their position within the mRNA. Taken together, the results show that variation in codon use is a major determinant of mRNA levels in T. brucei.