Frequent GU wobble pairings reduce translation efficiency in Plasmodium falciparum

Abstract

Plasmodium falciparum genome has 81% A+T content. This nucleotide bias leads to extreme codon usage bias and culminates in frequent insertion of asparagine homorepeats in the proteome. Using recodonized GFP sequences, we show that codons decoded via G:U wobble pairing are suboptimal codons that are negatively associated to protein translation efficiency. Despite this, one third of all codons in the genome are GU wobble codons, suggesting that codon usage in P. falciparum has not been driven to maximize translation efficiency, but may have evolved as translational regulatory mechanism. Particularly, asparagine homorepeats are generally encoded by locally clustered GU wobble AAT codons, we demonstrated that this GU wobble-rich codon context is the determining factor that causes reduction of protein level. Moreover, insertion of clustered AAT codons also causes destabilization of the transcripts. Interestingly, more frequent asparagine homorepeats insertion is seen in single-exon genes, suggesting transcripts of these genes may have been programmed for rapid mRNA decay to compensate for the inefficiency of mRNA surveillance regulation on intronless genes. To our knowledge, this is the first study that addresses P. falciparum codon usage in vitro and provides new insights on translational regulation and genome evolution of this parasite.

Forces acting on codon bias in malaria parasites

“…falciparum 40 . In contrast, this work, and other key studies which preceded it 27 , 28 , 41 , indicate that synonymous mutations in highly expressed genes (including their stop sequences) are not simply neutral.…”

“…By studying asparigine homorepeats, Chan et al . were able to show directly that wobble pairings also reduce translation efficiency in Plasmodium falciparum 41 . Hence the fall in T3:C3 ratio in high expression genes (also reported by Chan et al .…”

“…Recent work by Chan et al . during the conduct of our study 41 explores this further, in particular at asparagine codons where AAT codons can only be decoded via a near-cognate tRNA predicted to bind only via a ‘wobble’ interaction 49 . Reading of codons by wobble binding in metazoans is associated with slower translation because of ribosomal stalling 11 .…”

“…In their recently published work, Chan et al . examined the top 5% expressed genes during the progression of the asexual cycle in a highly analogous approach to ours 41 .…”

Identification of long regulatory elements in the genome ofPlasmodium falciparumand other eukaryotes

“…The 3' UTR from P. falciparum HRPII was amplified from a PCC1 plasmid (82) and cloned using primers 1 and 2 and restriction sites SpeI and HindIII. The PfGFP was designed by codon optimization of the GFP using codon usage tables for P. falciparum 3D7 from Codon Usage Database http://www.kazusa.or.jp/codon, minimizing GU wobble pairings (83), and adding the 15-bp homology necessary for InFusion cloning. PfGFP was ordered as a DNA gBlocks R gene fragment from Integrated DNA Technologies and cloned using restriction sites HindIII and BamHI.…”