A Cryptic Targeting Signal Creates a Mitochondrial FEN1 Isoform with Tailed R-Loop Binding Properties

A growing number of DNA transacting proteins is found in the nucleus and in mitochondria, including the DNA repair and replication protein Flap endonuclease 1, FEN1. Here we show a truncated FEN1 isoform is generated by alternative translation initiation, exposing a mitochondrial targeting signal. The shortened form of FEN1, which we term FENMIT, localizes to mitochondria, based on import into isolated organelles, immunocytochemistry and subcellular fractionation. In vitro FENMIT binds to flap structures containing a 5′ RNA flap, and prefers such substrates to single-stranded RNA. FENMIT can also bind to R-loops, and to a lesser extent to D-loops. Exposing human cells to ethidium bromide results in the generation of RNA/DNA hybrids near the origin of mitochondrial DNA replication. FENMIT is recruited to the DNA under these conditions, and is released by RNase treatment. Moreover, high levels of recombinant FENMIT expression inhibit mtDNA replication, following ethidium bromide treatment. These findings suggest FENMIT interacts with RNA/DNA hybrids in mitochondrial DNA, such as those found at the origin of replication.

目前在细胞核与线粒体中已发现越来越多的反式作用DNA蛋白（DNA transacting proteins），其中涵盖DNA修复与复制相关的瓣状核酸内切酶1（Flap endonuclease 1, FEN1）。本研究证实，选择性翻译起始（alternative translation initiation）可产生截短型FEN1同工型，该过程会暴露一段线粒体靶向信号（mitochondrial targeting signal）。我们将该截短形式的FEN1命名为FENMIT，通过离体细胞器导入实验、免疫细胞化学（immunocytochemistry）及亚细胞分级分离（subcellular fractionation）实验验证，FENMIT定位于线粒体。体外实验显示，FENMIT可结合带有5'端RNA瓣的瓣状结构，且相较于单链RNA（single-stranded RNA），其对这类底物展现出更高的结合偏好性。此外，FENMIT还能结合R环（R-loops），对D环（D-loops）的结合能力相对较弱。将人类细胞暴露于溴化乙锭（ethidium bromide）环境中时，会在线粒体DNA复制起点附近生成RNA/DNA杂交体（RNA/DNA hybrids）。在此条件下，FENMIT会被招募至DNA位点，经核糖核酸酶（RNase）处理后可被释放。进一步研究发现，高表达重组FENMIT（recombinant FENMIT）会抑制溴化乙锭处理后的线粒体DNA（mtDNA）复制。上述研究结果表明，FENMIT可与线粒体DNA中的RNA/DNA杂交体相互作用，例如复制起点处存在的这类杂交体。