Evidence for a novel overlapping coding sequence in POLG initiated at a CUG start codon

Yousuf A Khan,Irwin Jungreis,Andrew E Firth,Jonathan M Mudge,Manolis Kellis,Jyoti S Choudhary,James C Wright

doi:10.1186/s12863-020-0828-7

Abstract

BackgroundPOLG, located on nuclear chromosome 15, encodes the DNA polymerase γ(Pol γ). Pol γ is responsible for the replication and repair of mitochondrial DNA (mtDNA). Pol γ is the only DNA polymerase found in mitochondria for most animal cells. Mutations in POLG are the most common single-gene cause of diseases of mitochondria and have been mapped over the coding region of the POLG ORF.ResultsUsing PhyloCSF to survey alternative reading frames, we found a conserved coding signature in an alternative frame in exons 2 and 3 of POLG, herein referred to as ORF-Y that arose de novo in placental mammals. Using the synplot2 program, synonymous site conservation was found among mammals in the region of the POLG ORF that is overlapped by ORF-Y. Ribosome profiling data revealed that ORF-Y is translated and that initiation likely occurs at a CUG codon. Inspection of an alignment of mammalian sequences containing ORF-Y revealed that the CUG codon has a strong initiation context and that a well-conserved predicted RNA stem-loop begins 14 nucleotides downstream. Such features are associated with enhanced initiation at near-cognate non-AUG codons. Reanalysis of the Kim et al. (2014) draft human proteome dataset yielded two unique peptides that map unambiguously to ORF-Y. An additional conserved uORF, herein referred to as ORF-Z, was also found in exon 2 of POLG. Lastly, we surveyed Clinvar variants that are synonymous with respect to the POLG ORF and found that most of these variants cause amino acid changes in ORF-Y or ORF-Z.ConclusionsWe provide evidence for a novel coding sequence, ORF-Y, that overlaps the POLG ORF. Ribosome profiling and mass spectrometry data show that ORF-Y is expressed. PhyloCSF and synplot2 analysis show that ORF-Y is subject to strong purifying selection. An abundance of disease-correlated mutations that map to exons 2 and 3 of POLG but also affect ORF-Y provides potential clinical significance to this finding.

Highlights

DNA polymerase subunit gamma (POLG), located on nuclear chromosome 15, encodes the DNA polymerase γ(Pol γ)
The mitochondrial genome in humans is a circular DNA that encodes 13 proteins related to the function of the electron transport chain (ETC), 22 Transfer RNA (tRNA), and 2 Ribosomal RNA (rRNA) [2]. mitochondrial DNA (mtDNA) is replicated by a complex of Pol γ, a ssDNA binding protein, the Twinkle mtDNA helicase, topoisomerases, and RNaseH activity [3]
To find novel coding regions we had computed PhyloCSF scores for every codon in the human genome in each of six reading frames, used a hidden Markov model to find potential coding intervals, and screened out intervals overlapping known coding or pseudogenic regions in the same frame or the antisense frame, leaving us with approximately 70,000 PhyloCSF Candidate Coding Regions (PCCRs), which were prioritized by a machine learning algorithm and the first 1000 examined by expert manual annotators

Summary

Introduction

POLG, located on nuclear chromosome 15, encodes the DNA polymerase γ(Pol γ). Pol γ is responsible for the replication and repair of mitochondrial DNA (mtDNA). Pol γ is the only DNA polymerase found in mitochondria for most animal cells. Mutations in POLG are the most common single-gene cause of diseases of mitochondria and have been mapped over the coding region of the POLG ORF. The mitochondrial genome in humans is a circular DNA that encodes 13 proteins related to the function of the ETC, 22 tRNAs, and 2 rRNAs [2]. Mutations have been mapped across the entire coding region of POLG from exons 2 to 23 The underlying mechanism for the progression of these diseases is typically related to a depletion of mtDNA or mutation of mtDNA due to a defective Pol γ [8]. There is currently a dearth of therapies for disorders caused by POLG mutations despite how widely it influences the population [7]

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Conclusion