Alternative non-canonical translation initiation codons are used to synthesise novel isoforms of the transcription factor GATAD1

Abstract

Alternative translation initiation from upstream non-AUG codons contributes towards the diversity of the eukaryotic proteome; protein isoforms with varying N-terminal extensions can be generated from one mRNA transcript. Investigations are being carried out in order to elucidate how N-terminal extensions affect subcellular localisation, binding partners and thus the function of a protein as well as how the choice of alternative initiation codon (AIC) is regulated.
This thesis is focussed on GATAD1 (GATA Zinc Finger Domain-Containing 1), which is a ubiquitously expressed transcription factor, forming part of a transcriptionally repressive histone demethylase complex. GATAD1 regulates the expression of specific genes by forming an indirect interaction with the activating trimethyl marker of lysine four on histone three (H3K4me3); this interaction is made through a lysine demethylase chromatin ‘reader’, KDM5A (Jarid1A). GATAD1 is also involved in retinal development and heart disease, whereby a single mutation in the gene is the cause of dilated cardiomyopathy (DCM).

The GATAD1 mRNA transcript can be translated at alternative translation initiation codons resulting in the synthesis of three protein isoforms. The two isoforms with N-terminal extensions are initiated from a CUG and an unusual AUU codon. CRISPR genome editing has been used to tag genomic GATAD1, confirming endogenous expression of all three isoforms. Translation from the AICs is regulated by various factors, including eukaryotic initiation factors (eIFs) 1, 1A and 5, the context and position of the AICs, as well as secondary structure downstream of the AUU codon. Cell type and stresses such as hypoxia also influence the use of each GATAD1 AIC.

Although all three GATAD1 isoforms complex with KDM5A, it has been observed that the extended isoforms have a greater tendency to remain in the cytoplasm, potentially forming part of a cytoplasmic demethylase complex, whilst the annotated protein functions as a nuclear transcription factor.

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ORCID for Mark Coldwell: orcid.org/0000-0002-6243-3886

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