Expanded Polyglycine Protein Aggregates Alter the RNA Splicing Profile into a Premature State Partially via...

The GGC trinucleotide repeat expansion within NOTCH2NLC is the major genetic cause of neuronal intranuclear inclusion disease (NIID), and the encoded toxic protein containing the corresponding polyglycine (polyG) stretch, uN2CpolyG, is likely the main pathogenic molecule; however, the exact underlying pathogenic mechanisms of NIID remain unclear. Using fluorescence-activated cell sorting (FACS)-based enrichment of uN2CpolyG aggregates and proteomic analyses, we identified several key splicing factors, especially SF3B4, as proteins sequestered within the uN2CpolyG aggregates. A reanalysis of previously published transcriptome data revealed that brain tissue from NIID mice and human neural progenitor cells expressing uN2CpolyG exhibited altered global splicing profiles that resembled those of immature, developing neurons. Interestingly, knockdown of SF3B4 in control cells expressing the non-pathogenic short polyG protein (uN2Cpoly17G) partially recapitulated these changes, whereas SF3B4 knockdown in cells expressing the pathogenic long polyG protein (uN2Cpoly98G) partially attenuated these alterations, suggesting that the sequestration of SF3B4 by uN2CpolyG may contribute to the splicing alterations in NIID by mimicking SF3B4 loss-of-function. Conversely, SF3B4 knockdown also enhanced uN2Cpoly98G aggregation, suggesting a positive feedback loop that may exacerbate protein aggregation and splicing defects during disease progression. Taken together, our study suggests that polyG protein aggregates in NIID may shift the global RNA splicing profile toward an immature state, potentially through the sequestration of key splicing factors, such as SF3B4, thereby identifying new avenues for understanding the pathogenic mechanisms underlying NIID.