Recoding and reassignment in protists

Abstract

During mRNA translation the ribosome reads each codon (nucleotide triplet) with a specific meaning. The standard genetic code comprises 61 sense-codons for specifying the 20 standard amino acids during elongation and three anti-sense codons which signal termination. While variations to the standard rules of genetic decoding are widely acknowledged, recent advances in next generation sequencing techniques have provided a wealth of new examples across many species. In this thesis, I provide evidence of novel decoding mechanisms in protists, as identified through bioinformatics analysis. To begin with I analysed the genomes of two ciliate species, Euplotes crassus and E. focardii. In combination with the analysis of E. crassus transcriptome using ribosome profiling, I determined over 1,700 cases of ribosomal frameshifting (22% of genes analysed) in E. crassus. I identified 47 codons upstream of a stop signal which directs the ribosome to either the +1 or +2 reading frames. Termination only occurs in the context of the poly-A tail. In addition I analysed the transcriptomes of over 200 diverse protist species for the protein ornithine decarboxylase antizyme, a key negative regulator of cellular polyamine synthesis. The synthesis of this protein usually requires a +1 ribosomal frameshift at the end of the first open reading frame. In this study I identified a novel mechanism of stop codon readthrough to regulate antizyme production in dinoflagellates and single ORF sequences from other protist phyla. Further I analysed transcriptomes of diverse ciliate organisms to characterize stop codon reassignments in their genetic codes. In addition to finding novel stop codon reassignments, I identified an organism, Condylostoma magnum where all three stop codons TAA, TAG & TGA have been reassigned to sense codons. All three stop codons are enriched at the expected positions of translation termination sites which occur at a short distance from the 3’ poly-A tail.