Sequence-dependent mechanochemical coupling of helicase translocation and unwinding at single-nucleotide resolution.

We used single-molecule picometer-resolution nanopore tweezers (SPRNT) to resolve the millisecond single-nucleotide steps of superfamily 1 helicase PcrA as it translocates on, or unwinds, several kilobase-long DNA molecules. We recorded more than two million enzyme steps under various assisting and opposing forces in diverse adenosine tri- and diphosphate conditions to comprehensively explore the mechanochemistry of PcrA motion. Forces applied in SPRNT mimic forces and physical barriers PcrA experiences in vivo, such as when the helicase encounters bound proteins or duplex DNA. We show how PcrA's kinetics change with such stimuli. SPRNT allows for direct association of the underlying DNA sequence with observed enzyme kinetics. Our data reveal that the underlying DNA sequence passing through the helicase strongly influences the kinetics during translocation and unwinding. Surprisingly, unwinding kinetics are not solely dominated by the base pairs being unwound. Instead, the sequence of the single-stranded DNA on which the PcrA walks determines much of the kinetics of unwinding.

Nuclear export and translation of circular repeat-containing intronic RNA in C9ORF72-ALS/FTD.

C9ORF72 hexanucleotide GGGGCC repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-containing RNA mediates toxicity through nuclear granules and dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG translation. However, it remains unclear how the intron-localized repeats are exported and translated in the cytoplasm. We use single molecule imaging approach to examine the molecular identity and spatiotemporal dynamics of the repeat RNA. We demonstrate that the spliced intron with G-rich repeats is stabilized in a circular form due to defective lariat debranching. The spliced circular intron, instead of pre-mRNA, serves as the translation template. The NXF1-NXT1 pathway plays an important role in the nuclear export of the circular intron and modulates toxic DPR production. This study reveals an uncharacterized disease-causing RNA species mediated by repeat expansion and demonstrates the importance of RNA spatial localization to understand disease etiology.