AAGGG repeat expansions trigger RFC1-independent synaptic dysregulation in human CANVAS neurons.

Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a recessively inherited neurodegenerative disorder caused by intronic biallelic, nonreference CCCTT/AAGGG repeat expansions within RFC1. To investigate how these repeats cause disease, we generated patient induced pluripotent stem cell-derived neurons (iNeurons). CCCTT/AAGGG repeat expansions do not alter neuronal RFC1 splicing, expression, or DNA repair pathway function. In reporter assays, AAGGG repeats are translated into pentapeptide repeat proteins. However, these proteins and repeat RNA foci were not detected in iNeurons, and overexpression of these repeats failed to induce neuronal toxicity. CANVAS iNeurons exhibit defects in neuronal development and diminished synaptic connectivity that is rescued by CRISPR deletion of a single expanded AAGGG allele. These deficits were neither replicated by RFC1 knockdown in control iNeurons nor rescued by RFC1 reprovision in CANVAS iNeurons. These findings support a repeat-dependent but RFC1 protein-independent cause of neuronal dysfunction in CANVAS, with implications for therapeutic development in this currently untreatable condition.

TDP43 autoregulation gives rise to shortened isoforms that are tightly controlled by both transcriptional and post-translational mechanisms.

The nuclear RNA-binding protein TDP43 is integrally involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Previous studies uncovered N-terminal TDP43 isoforms that are predominantly cytosolic in localization, highly prone to aggregation, and enriched in susceptible spinal motor neurons. In healthy cells, however, these shortened (s)TDP43 isoforms are difficult to detect in comparison to full-length (fl)TDP43, raising questions regarding their origin and selective regulation. Here, we show that sTDP43 is created as a byproduct of TDP43 autoregulation and cleared by nonsense mediated RNA decay (NMD). The sTDP43-encoding transcripts that escape NMD can lead to toxicity but are rapidly degraded post-translationally. Circumventing these regulatory mechanisms by overexpressing sTDP43 results in neurodegeneration in vitro and in vivo via N-terminal oligomerization and impairment of flTDP43 splicing activity, in addition to RNA binding-dependent gain-of-function toxicity. Collectively, these studies highlight endogenous mechanisms that tightly regulate sTDP43 expression and provide insight into the consequences of aberrant sTDP43 accumulation in disease.

Intracellular RNA and DNA tracking by uridine-rich internal loop tagging with fluorogenic bPNA.

The most widely used method for intracellular RNA fluorescence labeling is MS2 labeling, which generally relies on the use of multiple protein labels targeted to multiple RNA (MS2) hairpin structures installed on the RNA of interest (ROI). While effective and conveniently applied in cell biology labs, the protein labels add significant mass to the bound RNA, which potentially impacts steric accessibility and native RNA biology. We have previously demonstrated that internal, genetically encoded, uridine-rich internal loops (URILs) comprised of four contiguous UU pairs (8 nt) in RNA may be targeted with minimal structural perturbation by triplex hybridization with 1 kD bifacial peptide nucleic acids (bPNAs). A URIL-targeting strategy for RNA and DNA tracking would avoid the use of cumbersome protein fusion labels and minimize structural alterations to the RNA of interest. Here we show that URIL-targeting fluorogenic bPNA probes in cell media can penetrate cell membranes and effectively label RNAs and RNPs in fixed and live cells. This method, which we call fluorogenic U-rich internal loop (FLURIL) tagging, was internally validated through the use of RNAs bearing both URIL and MS2 labeling sites. Notably, a direct comparison of CRISPR-dCas labeled genomic loci in live U2OS cells revealed that FLURIL-tagged gRNA yielded loci with signal to background up to 7X greater than loci targeted by guide RNA modified with an array of eight MS2 hairpins. Together, these data show that FLURIL tagging provides a versatile scope of intracellular RNA and DNA tracking while maintaining a light molecular footprint and compatibility with existing methods.