New research published in Nucleic Acids Research shows that the mitochondrial protein Twinkle has unexpected RNA-related activities that may help explain disease mechanisms in thymidine kinase 2 deficiency (TK2d) and related mitochondrial disorders.
Twinkle is essential for copying mitochondrial DNA, the genetic material that powers cells. When this process goes wrong, as it does in TK2d, cells struggle to make energy, leading to muscle weakness, breathing problems and other serious symptoms. The new findings suggest that Twinkle’s balance of actions, not just its ability to unwind DNA, matters for mitochondrial health.
Twinkle has long been known as the only helicase that unwinds mitochondrial DNA so it can be copied. Scientists now report that Twinkle also binds tightly to RNA and to RNA:DNA hybrids, with binding strengths in the low nanomolar range, similar to its grip on DNA. This means Twinkle can connect RNA strands to DNA strands and even help swap strands between them. These actions are unusual for a protein whose main job is DNA replication.
This study shows that Twinkle can unwind mixed RNA:DNA structures, but only when it moves along a DNA strand, not an RNA strand. This detail matters because RNA:DNA hybrids, sometimes called R-loops, can be helpful or harmful. In healthy cells, they may help restart stalled DNA replication. If they persist too long, they can damage mitochondrial DNA. For patients with TK2d, whose mitochondria are already under stress, this balance may be especially fragile.
“Ultimately, our findings have implications for human health and disease,” said the authors of this study.
Read more about the causes and risk factors of TK2d
Researchers also found that Twinkle can actively join RNA to DNA through rapid annealing and strand exchange. These reactions were hundreds of times faster with Twinkle than without it. In lab experiments, Twinkle even helped an RNA primer restart DNA replication after it stalled, allowing the mitochondrial DNA polymerase to resume copying. This offers a possible explanation for how mitochondria cope with damage when resources are limited, as in TK2d.
Another mitochondrial protein, mtSSB, was shown to rein in these RNA-related activities. At higher levels, mtSSB blocked Twinkle from forming RNA:DNA hybrids, suggesting a built-in control system. If this control fails, harmful hybrids could accumulate, further damaging mitochondrial DNA in diseases like TK2d.
Finally, a disease-linked Twinkle variant called W315L highlighted the risks of imbalance. This variant struggled to support normal DNA replication but still promoted RNA:DNA strand exchange. For patients, this supports the idea that mitochondrial disease may arise not only from lost activity, but from the wrong activity happening at the wrong time. Understanding these details could eventually lead to treatments that restore balance and protect mitochondrial DNA in TK2d and beyond.
Sign up here to get the latest news, perspectives, and information about TK2d sent directly to your inbox. Registration is free and only takes a minute.