SheqLab

And that's a wrap! I hope you've enjoyed this thread and give our preprint a read. Let us know what you think! tinyurl.com/45ds5ccs

Taken together, our data allow us to propose the first-ever model for the molecular architecture of a single-locus subnuclear compartment. This dramatically expands our understanding of the factory's functional scope, and how its disruption may contribute to DCM.

QKI and SAFB recruitment required both RBM20 and TTN RNA, but not each other—evoking a scaffold-client model seen in other nuclear bodies. Oddly, SAFB knockdown opposed the effects of RBM20 loss on many TID-localized genes' expression. Maybe these factors act in opposition?

Finally, we used O-MAP to probe the TTN factory proteome. This remarkable compartment appears to integrate RNA biogenesis with chromatin compartmentalization, recruiting tons of exciting chromatin regulators (cohesin; SAFB) and RBPs (QKI; SRSFs; ELAVL1).

...And that is *exactly* what we observe!! Removing the TTN promoter in ∆RBM20 cells attenuates ~80% of all splicing defects, partially or fully restoring them to WT splicing patterns. Perhaps similar RNA toxicity contributes to other spliceopathies?

Intriguingly, the splicing of TTN-proximal RNAs was especially sensitive to RBM20 loss, even though very few of these transcripts are known RBM20-targets. In fact, in ∆RBM20 cells, fewer than 5% of dysregulated splicing events were directly regulated by RBM20. What gives?

We next characterized the TTN factory transcriptome, revealing hundreds of RNAs that specifically localize near nascent TTN in WT and ∆RBM20 cells. Almost none of these RNAs are encoded in TTN-proximal DNA loci, suggesting that they're localized post-transcriptionally.

We believe this chromosomal "clique" still forms in ∆RBM20 cells but is slightly weakened—inducing mild, though pervasive changes in gene expression. Awesome work from @NucleomeHunter's lab further showed that the TTN TID clique is cardiac-specific and developmentally regulated.

We began by probing the factory's genomic interactions, which revealed a hub of Mb-scale, TTN-interacting chromatin domains in both cis (CIDs) and trans (TIDs). Strikingly, in ∆RBM20 cells, these TTN RNA–chromatin contacts completely disappear!

This approach yielded exceptionally precise biotinylation in both WT and ∆RBM20 cardiomyocytes: just two biotin foci—corresponding to the two alleles of TTN. In the context of two meters of folded DNA, O-MAP can home in and affinity-tag a single-locus nuclear compartment.