RESUMO
C9ORF72 hexanucleotide repeat expansion is the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One pathogenic mechanism is the accumulation of toxic dipeptide repeat (DPR) proteins like poly-GA, GP and GR, produced by the noncanonical translation of the expanded RNA repeats. However, how different DPRs are synthesized remains elusive. Here, we use single-molecule imaging techniques to directly measure the translation dynamics of different DPRs. Besides initiation, translation elongation rates vary drastically between different frames, with GP slower than GA and GR the slowest. We directly visualize frameshift events using a two-color single-molecule translation assay. The repeat expansion enhances frameshifting, but the overall frequency is low. There is a higher chance of GR-to-GA shift than in the reversed direction. Finally, the ribosome-associated protein quality control (RQC) factors ZNF598 and Pelota modulate the translation dynamics, and the repeat RNA sequence is important for invoking the RQC pathway. This study reveals that multiple translation steps modulate the final DPR production. Understanding repeat RNA translation is critically important to decipher the DPR-mediated pathogenesis and identify potential therapeutic targets in C9ORF72-ALS/FTD.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Humanos , Demência Frontotemporal/genética , Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , RNA/genética , Imagem Individual de Molécula , Dipeptídeos , Proteínas de TransporteRESUMO
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.
Assuntos
Proteína C9orf72/genética , Núcleo Celular/metabolismo , Íntrons/genética , Biossíntese de Proteínas/genética , RNA/genética , Transporte Ativo do Núcleo Celular/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Proteína C9orf72/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/genética , Expansão das Repetições de DNA/genética , Dipeptídeos/genética , Dipeptídeos/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Predisposição Genética para Doença/genética , Células HEK293 , Humanos , Microscopia de Fluorescência , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genéticaRESUMO
The central dogma of molecular biology reaches a crescendo at its final step: the translation of an mRNA into its corresponding protein product. This process is highly regulated both spatially and temporally, requiring techniques to interrogate the subcellular translational status of mRNAs in both living and fixed cells. Single-molecule imaging of nascent peptides (SINAPs) and related techniques allow us to study this fundamental process for single mRNAs in live cells. These techniques enable researchers to address previously intractable questions in the central dogma, such as the origin of stochastic translational control and the role of local translation in highly polarized cells. In this review, we present the methodology and the theoretical framework for conducting studies using SINAPs in both established cell lines and primary cultured neurons.
