Pathogenic mutations reveal a role of RECQ4 in mitochondrial RNA:DNA hybrid formation and resolution

Abstract

The synthesis of mitochondrial DNA (mtDNA) is a complex process that involves the formation and resolution of unusual nucleic acid structures, such as RNA:DNA hybrids. However, little is known about the enzymes that regulate these processes. RECQ4 is a DNA replication factor important for mtDNA maintenance, and here, we unveil a role of human RECQ4 in regulating the formation and resolution of mitochondrial RNA:DNA hybrids. Mitochondrial membrane protein p32 can block mtDNA synthesis by restricting RECQ4 mitochondrial localization via protein–protein interaction. We found that the interaction with p32 was disrupted not only by the previously reported cancer-associated RECQ4 mutation, del(A420-A463), but also by a clinical mutation of the adjacent residue, P466L. Surprisingly, although P466L mutant was present in the mitochondria at greater levels, unlike del(A420-A463) mutant, it failed to enhance mtDNA synthesis due to the accumulation of RNA:DNA hybrids throughout the mtDNA. Biochemical analysis revealed that P466L mutation enhanced RECQ4 annealing activity to generate RNA:DNA hybrids at the same time reduced its unwinding activity to resolve this structure. Hence, P466L mutation led to a reduced efficiency in completing mtDNA synthesis due to unresolved RNA:DNA hybrids across mtDNA.

Molecular Mechanisms of the RECQ4 Pathogenic Mutations

“…Studies using RECQ4 disease mutations reveal a role of RECQ4 in mitochondrial biogenesis. For example, RECQ4 is involved in mitochondrial DNA (mtDNA) synthesis and maintenance, and abnormal mtDNA levels were observed in the RECQ4 mutant cells ( Croteau et al, 2012a ; De et al, 2012 ; Wang et al, 2014 ; Chang et al, 2020 ). The changes in mtDNA copy numbers and their contents correlate with the mitochondrial dysfunction phenotypes in human cells ( Croteau et al, 2012a ; De et al, 2012 ; Wang et al, 2014 ; Chang et al, 2020 ).…”

“…For example, RECQ4 is involved in mitochondrial DNA (mtDNA) synthesis and maintenance, and abnormal mtDNA levels were observed in the RECQ4 mutant cells ( Croteau et al, 2012a ; De et al, 2012 ; Wang et al, 2014 ; Chang et al, 2020 ). The changes in mtDNA copy numbers and their contents correlate with the mitochondrial dysfunction phenotypes in human cells ( Croteau et al, 2012a ; De et al, 2012 ; Wang et al, 2014 ; Chang et al, 2020 ). RECQ4 interacts with mitochondrial replication factors, including TWINKLE/PEO1, TFAM and DNA polymerase γ ( Croteau et al, 2012a ; Wang et al, 2014 ).…”

“…As a consequence, cells expressing RECQ4 del(Ala420-Ala463) mutant protein show altered oxidative phosphorylation efficiency for mitochondrial-dependent ATP production ( Croteau et al, 2012a ; Wang et al, 2014 ; Kumari et al, 2016 ). Interestingly, an adjacent RAPADILINO mutation Pro466Leu also impairs RECQ4 interaction with p32, leading to the accumulation of the mutant protein in the mitochondria ( Chang et al, 2020 ). However, unlike cells expressing the del(Ala420-Ala463) mutant proteins, Pro466Leu expression does not accelerate mtDNA synthesis despite the fact that the point mutation also enhances RECQ4-TWINKLE interaction ( Chang et al, 2020 ).…”

“…Interestingly, an adjacent RAPADILINO mutation Pro466Leu also impairs RECQ4 interaction with p32, leading to the accumulation of the mutant protein in the mitochondria ( Chang et al, 2020 ). However, unlike cells expressing the del(Ala420-Ala463) mutant proteins, Pro466Leu expression does not accelerate mtDNA synthesis despite the fact that the point mutation also enhances RECQ4-TWINKLE interaction ( Chang et al, 2020 ). These observations highlight the heterogeneity in mitochondrial dysfunction observed in different RECQ4 patient cell lines ( Croteau et al, 2012a ).…”

“…Structural analysis located a second R ECQ 4 z inc- b inding d omain (R4ZBD; Figure 1 ) at the C-terminus of RECQ4 between residues 836 and 1,044, but R4ZBD does not play a significant role in DNA binding or ATPase activities ( Kaiser et al, 2017 ). The nucleic acid binding by the N-terminal fragment of RECQ4 contributes to its strand annealing and strand exchange activities ( Xu and Liu, 2009 ; Keller et al, 2014 ), providing an explanation for why pathogenic mutations within the RECQ4 N-terminus affect its annealing and strand exchange activities ( Chang et al, 2020 ). RECQ4 N-terminus also shows high affinities toward unusual DNA structures, such as guanine quadruplex and RNA:DNA hybrids ( Keller et al, 2014 ; Chang et al, 2020 ), supporting additional roles of RECQ4 N-terminus in nucleic acid metabolism, which is altered by the pathogenic RECQ4 Pro466Leu mutation.…”