1. Determine the molecular mechanisms of repeat RNA metabolisms and RAN translation

Nucleotide repeat expansions underlie a heterogeneous groups of neurological and neuromuscular disorders with various disease mechanisms depending on the gene function, structure and location of the expansion. Hexanucleotide repeat expansion in the C9orf72 gene is the most frequent inherited cause of both ALS and FTD. A leading hypothesis for disease mechanism is gain of toxicity from the expanded repeats, which are transcribed in both sense and antisense directions and give rise to distinct sets of intranuclear RNA foci. This could sequester certain RNA-binding proteins and lead to their loss of function. An alternative but not mutually exclusive mechanism is the aberrant accumulation of dipeptide repeat proteins produced by repeat-associated non-ATG (RAN) translation in all six reading frames (poly-GA, poly-GR, poly-PA, poly-PR and poly-PG) of both strands. A third hypothesis is the expansions cause gene silencing and lead to haploid insufficiency of C9orf72 protein. Till today, there is no established evidence supporting what is the main factor driving the disease pathogenesis. Our research goal is to use biochemical, proteomic, genomic and screening approaches to decipher the various mechanisms of repeat expansion mediated toxicity and advance therapeutic target development, especially by applying RNA Biology knowledge and technologies.

1.1 Determine the molecular mechanisms of RAN translation

Repeat expansions do not follow the canonical rules of ATG-dependent translation initiation and can generate a series of aberrant repeat proteins in multiple reading frames. This phenomenon has been identified in several repeat expansion disorders, including myotonic dystrophy (CAG repeats), spinocerebellar ataxia type 8 (CAG repeats), Fragile X-associated tremor/ataxia syndrome (CGG repeats), Huntington’s disease (CAG repeats), and now C9orf72-ALS/FTD. Despite the evidence of dipeptide accumulation in patients and their potential toxicity on neurons, the mechanisms of how RAN translation occurs and is regulated remain largely unknown.  Identification of specific modifiers of RAN translation will provide tools to distinguish the RNA- versus dipeptides-mediated toxicity and develop drug targets to reduce dipeptide production as a therapeutic strategy. Furthermore, it is not known what is the relative translational rate of different dipeptides from different reading frames, whether there is frame shift on the repeats during translation elongation, et al. Understanding the molecular mechanisms of RAN translation is essential to understand their toxic contribution to the neurodegeneration, and also addresses an interesting basic biological question of normal non-canonical translation process that could have physiological functions in other settings.

  • Identification of genetic modifiers of RAN translation using CRISPR/Cas9 genomic screening.
  • Single molecule imaging of RNAs and nascent peptides (SINAPS) to dissect RAN translation mechanisms at single molecule level in live cells.
  • Determine how RAN translation is regulated during stress and aging process.

1.2 Investigate the repeat RNA metabolism

Bi-directional transcription of the C9ORF72 repeats leads to the formation of both sense (GGGGCC)n and antisense (CCCCGG)n RNA repeats. On one hand, they form nuclear granules. On the other hand, the repeat RNAs are subjected to RAN translation in cytoplasm. The sub-cellular distribution and molecular properties of the different repeats for different functions remain mystery. The molecular insights obtained through this research will help understand the disease mechanisms and guide the therapeutic design.

  • Single molecule imaging of RNAs to determine the dynamics of RNA repeats in live cells.
  • Phase separation of repeat RNA and the effects on endogenous RNP granules.

2. Decipher the global RNA metabolism dysregulation in ALS/FTD 

Many disease causative genes in ALS and FTD are linked to RNA regulation, including C9ORF72 repeat expansion and RNA-binding proteins (RBPs), such as TDP-43 and FUS/TLS. Besides causative mutations found in familial ALS, the pathology of RBPs is widely found in sporadic ALS and FTD. Understanding how the endogenous RNA metabolism pathways are affected by the mutated or pathological RBPs and expanded repeats will likely provide novel insights on biomarker and therapeutic targets development. Neurons have extremely complex RNA metabolism and highly specialized RNA processing pathways, probably due to their highly complex morphologies and functions. We are interested in deciphering the global RNA metabolism dysregulation by high-throughput sequencing techniques, including splicing, translation, modification, and the connection to epigenetic regulation via ncRNAs.

3. Identify disease-modifying genes by CRISPR screening in human iPSC-derived neurons

Genetic screenings carried out on model organisms such as yeast, flies and nematodes have played important roles in understanding gene functions and the pathogenic mechanisms induced by disease-causative mutations, which facilitate the development of novel therapeutic targets. We have established the cutting-edge CRISPR-Cas9 screening platform to screen for modifiers directly in human cells and iPSC-derived neurons. We develop assays to screen for modifiers of specific molecular pathways (RNA processing, protein homeostasis), as well as ones that can inhibit mutant gene-induced toxicity and improve neuron survival. Identification of druggable gene products will facilitate treatment development of neurodegenerative diseases.