The human DBR1 interactome reveals coupling between intron lariat turnover, pre-mRNA splicing, and RNA quality control pathways.

Pre-mRNA splicing produces intron lariats that must be cleaved at their internal 2-5' phosphodiester bond by the debranching endonuclease DBR1. While human DBR1 (hDBR1) is established as the lariat debranching enzyme, how it interfaces with broader RNA metabolic pathways is less clear. Using chemical inhibition of splicing, we show that DBR1 expression correlates with splicing activity. We then mapped the hDBR1 interactome by immunopurification coupled to mass spectrometry using complementary gel-based and on-bead workflows. hDBR1 associates with the spliceosome and intron-turnover factors, and with RNA quality-control proteins, including UPF1, XRN2, and the RNA helicase DHX29. RNase A treatment identifies an RNA-dependent subnetwork enriched for stress-granule proteins and hnRNPs, linking hDBR1 to RNA surveillance during stress. Comparison with BioGRID indicates that most detected associations were not previously reported. Finally, phosphoproteomic profiling reveals multiple hDBR1 phosphorylation sites, including four residues preferentially detected after RNase treatment, suggesting regulatory modifications that may tune hDBR1 interactions or activity. Together, these data expand the functional landscape of hDBR1 across splicing, intron turnover, and RNA quality control.