Single Molecule Imaging of C9orf72 RNA and Repeat Associated Non-ATG Translation in Live Cells

Abstract

Amyotrophic lateral sclerosis (ALS) is currently an incurable disease caused by motor neuron degeneration. The most common cause of inheritable ALS is an expansion of hexanucleotide GGGGCC repeat in the C9orf72 gene. RNA containing the abnormally long GGGGCC repeats can form RNA foci, which may sequester essential RNA binding proteins. Alternatively, RNA containing repeats can be exported from nucleus and translated via repeat associated non-ATG (RAN) translation to generate different poly dipeptide repeats (DPR). The hexanucleotides are transcribed in both sense and antisense direction, which leads to two type of repetitive RNA and translation in six different reading frames. In patients the levels of DPR from each reading frame is different, which has been speculated resulting from difference in RAN translation efficiency. However, there is no direct evidence to support this claim. We used single-molecule imaging of nascent peptides reporter (SINAPs) to visualize and quantify the dynamics and heterogeneity of different dipeptides produced from C9orf72 repeat expansion. This approach allows us to measure the rate of translation of different reading frames in single molecule resolution. We found that RAN translation is noticeably less efficient than AUG dependent translation. However, different RAN reading frames have a similar rate of translation. Furthermore, we measured the rate of initiation and elongation speed through hexanucleotide repeats, which might explain the differences between the detected levels of the DPR's in patients. The molecular insights obtained through this study may help understanding the etiology of ALS and aid the therapeutic design.