Systematic generation and imaging of tandem repeats reveal base-pairing properties that promote RNA aggregation.

A common pathological feature of RNAs containing expanded repeat sequences is their propensity to aggregate in cells. While some repeat RNA aggregates have been shown to cause toxicity by sequestering RNA-binding proteins, the molecular mechanism of aggregation remains unclear. Here, we devised an efficient method to generate long tandem repeat DNAs de novo and applied it to systematically determine the sequence features underlying RNA aggregation. Live-cell imaging of repeat RNAs indicated that aggregation was promoted by multivalent RNA-RNA interactions via either canonical or noncanonical base pairs. While multiple runs of two consecutive base pairs were sufficient, longer runs of base pairs such as those formed by GGGGCC hexanucleotide repeats further enhanced aggregation. In summary, our study provides a unifying model for the molecular basis of repeat RNA aggregation and a generalizable approach for identifying the sequence and structural determinants underlying the distinct properties of repeat DNAs and RNAs.

C9orf72 arginine-rich dipeptide repeats inhibit UPF1-mediated RNA decay via translational repression.

Expansion of an intronic (GGGGCC)n repeat region within the C9orf72 gene is a main cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). A hallmark of c9ALS/FTD is the accumulation of misprocessed RNAs, which are often targets of cellular RNA surveillance. Here, we show that RNA decay mechanisms involving upstream frameshift 1 (UPF1), including nonsense-mediated decay (NMD), are inhibited in c9ALS/FTD brains and in cultured cells expressing either of two arginine-rich dipeptide repeats (R-DPRs), poly(GR) and poly(PR). Mechanistically, although R-DPRs cause the recruitment of UPF1 to stress granules, stress granule formation is independent of NMD inhibition. Instead, NMD inhibition is primarily a result from global translational repression caused by R-DPRs. Overexpression of UPF1, but none of its NMD-deficient mutants, enhanced the survival of neurons treated by R-DPRs, suggesting that R-DPRs cause neurotoxicity in part by inhibiting cellular RNA surveillance.