A genetic code expansion: investigation of UGA stop codon redefinition in selenoproteins

Baclaocos, Janinah

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

dc.check.chapterOfThesis	Chapter 2
dc.check.date	2022-09-17T08:43:58Z
dc.check.embargoformat	Apply the embargo to the e-thesis on CORA (If you have submitted an e-thesis and want to embargo it on CORA)	en
dc.check.info	Restricted to everyone for three years	en
dc.check.opt-out	Not applicable	en
dc.check.reason	This thesis contains data which has not yet been published	en
dc.contributor.advisor	Atkins, John F.	en
dc.contributor.advisor	Mackrill, John	en
dc.contributor.author	Baclaocos, Janinah
dc.contributor.funder	Science Foundation Ireland	en
dc.date.accessioned	2019-09-18T08:43:58Z
dc.date.issued	2019
dc.date.submitted	2019
dc.description.abstract	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.	en
dc.description.status	Not peer reviewed	en
dc.description.version	Accepted Version
dc.format.mimetype	application/pdf	en
dc.identifier.citation	Baclaocos, J. M. P. 2019. A genetic code expansion: investigation of UGA stop codon redefinition in selenoproteins. PhD Thesis, University College Cork.	en
dc.identifier.endpage	208	en
dc.identifier.uri	https://hdl.handle.net/10468/8558
dc.language.iso	en	en
dc.publisher	University College Cork	en
dc.rights	© 2019, Janinah Marie P. Baclaocos.	en
dc.rights.uri	http://creativecommons.org/licenses/by-nc-nd/3.0/	en
dc.subject	mRNA	en
dc.subject	Selenoprotein	en
dc.subject	Selenoprotein P	en
dc.subject	Selenium	en
dc.subject	Recoding	en
dc.subject	Ribosome	en
dc.subject	Translation	en
dc.subject	Genetic code	en
dc.subject	Frozen accident	en
dc.subject	Mollusc	en
dc.subject	Metazoa	en
dc.subject	Evolution	en
dc.subject	Ribosome profiling	en
dc.subject	Protein	en
dc.thesis.opt-out	false
dc.title	A genetic code expansion: investigation of UGA stop codon redefinition in selenoproteins	en
dc.type	Doctoral thesis	en
dc.type.qualificationlevel	Doctoral	en
dc.type.qualificationname	PhD	en
ucc.workflow.supervisor	j.atkins@ucc.ie