Restricted to everyone for three years. Restriction lift date: 2022-09-17T08:43:58Z
A genetic code expansion: investigation of UGA stop codon redefinition in selenoproteins
Full Text E-thesis
University College Cork
After the genetic code was deciphered in the 1960s, Francis Crick formulated the ‘frozen accident’ hypothesis (Crick, 1968) to describe the origins of the genetic code as universal and resistant to change or evolution. Co-incidentally, evidence of the dynamic nature of genetic decoding emerged through a series of experimental observations which presented various cases of exceptions from what were known as the standard rules of decoding. There is now prevalent understanding and evidence that the genetic code is constantly evolving, and it can be altered by various organisms with possible implications for entire genomes or specific mRNAs. The incorporation of the 21st amino-acid selenocysteine in selenoproteins in response to the UGA translation ‘terminator’ codon is an example of a gene-specific expansion of the code. This thesis will deal primarily with two unique cases of UGA recoding. The first case is the synthesis of selenophosphate synthetase 1 (SPS1) (Chapter 2) whereby an unknown amino acid is inserted in response to a UGA codon in the hymenopteran honeybee, Apis mellifera, which lacks the machinery for Sec incorporation. The various attempts to characterize the amino acid inserted at this position by novel methods are described. In Chapter 3, the first extensive evolutionary analysis of the selenium transporting protein, selenoprotein P (SELENOP) in invertebrates is described with focused characterization in the mollusc, Pacific oyster, Crassostrea gigas. This unique case presented an unprecedentedly high Sec content (46 Sec) in the C-terminal domain of its SELENOP highlighting an extreme case of deviation from the standard genetic code read-out. It was shown that a supplemented heterologous system, was able to facilitate translation of oyster SelenoP mRNA up to the third or fourth Sec codon position of the distal region but was inadequate to produce the full-length protein. Further, the Sec-dedicated protein factor, the oyster SECIS binding protein 2 (SBP2) was characterized and its potential tested for processive Sec-incorporation. Specific mRNA structures in the 3’UTR, termed Selenocysteine Insertion Sequence (SECIS), are essential for the recoding of UGA to specify selenocysteine instead of termination. While previously known multi-Sec codon SelenoP genes have two functionally distinct SECISes, the two in C. gigas showed no distinction in-vitro. In Chapter 4, in-vivo selenium regulation of selenoproteins in C.gigas was investigated by ribosome profiling. Total selenium levels in oyster tissues were found to increase up to 50-fold with supplementation, also resulting to an increase in mRNA abundance and translation. The translation of the full-length Pacific oyster SelenoP demonstrates an inefficient selenocysteine specification at UGA 1 (> 6%) and very high efficiency at the distal UGAs (UGAs 2 to 46). Additional genetic elements relevant to SelenoP translation include a leader ORF, and the RNA structure, termed Initiation Stem Loop (ISL) which were found to potentially modulate ribosome progression in a selenium-dependent manner. It was further validated that selenocysteines were metabolically incorporated in response to UGAs during the synthesis of oyster SELENOP as indicated by 75Selenium labelling experiments. These findings highlight the increasing understanding of the plasticity of the genetic code, as well as the ecological importance of selenium and its diverse utilization across species.
mRNA , Selenoprotein , Selenoprotein P , Selenium , Recoding , Ribosome , Translation , Genetic code , Frozen accident , Mollusc , Metazoa , Evolution , Ribosome profiling , Protein
Baclaocos, J. M. P. 2019. A genetic code expansion: investigation of UGA stop codon redefinition in selenoproteins. PhD Thesis, University College Cork.