The debranching enzyme Dbr1 regulates lariat turnover and intron splicing

• Published in: Nature communications 2024-05, Vol.15 (1), p.4617-13, Article 4617
• Main Authors: Buerer, Luke, Clark, Nathaniel E., Welch, Anastasia, Duan, Chaorui, Taggart, Allison J., Townley, Brittany A., Wang, Jing, Soemedi, Rachel, Rong, Stephen, Lin, Chien-Ling, Zeng, Yi, Katolik, Adam, Staley, Jonathan P., Damha, Masad J., Mosammaparast, Nima, Fairbrother, William G.
• Format: Article
• Language: English
• Subjects: 45; 45/23; 45/91; 49; 49/40; 631/337; 631/337/1645/1792; 631/45; 631/45/612/1230; 631/45/612/1242; Catalysis; Depletion; Dwell time; Enzymes; Exon skipping; Exons - genetics; HEK293 Cells; HeLa Cells; Humanities and Social Sciences; Humans; Immunoprecipitation; Introns; Introns - genetics; Mass spectrometry; Mass spectroscopy; multidisciplinary; Protein folding; RNA Nucleotidyltransferases - genetics; RNA Nucleotidyltransferases - metabolism; RNA Splice Sites; RNA Splicing; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Science; Science (multidisciplinary); Spliceosomes - metabolism; Splicing; Substrates
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-48696-1

The majority of genic transcription is intronic. Introns are removed by splicing as branched lariat RNAs which require rapid recycling. The branch site is recognized during splicing catalysis and later debranched by Dbr1 in the rate-limiting step of lariat turnover. Through generation of a viable DBR1 knockout cell line, we find the predominantly nuclear Dbr1 enzyme to encode the sole debranching activity in human cells. Dbr1 preferentially debranches substrates that contain canonical U2 binding motifs, suggesting that branchsites discovered through sequencing do not necessarily represent those favored by the spliceosome. We find that Dbr1 also exhibits specificity for particular 5’ splice site sequences. We identify Dbr1 interactors through co-immunoprecipitation mass spectrometry. We present a mechanistic model for Dbr1 recruitment to the branchpoint through the intron-binding protein AQR. In addition to a 20-fold increase in lariats, Dbr1 depletion increases exon skipping. Using ADAR fusions to timestamp lariats, we demonstrate a defect in spliceosome recycling. In the absence of Dbr1, spliceosomal components remain associated with the lariat for a longer period of time. As splicing is co-transcriptional, slower recycling increases the likelihood that downstream exons will be available for exon skipping. Dbr1 exhibits debranching specificity and effect on splicing. Here the authors combine co-immunoprecipitation, RNA binding and lariat analysis and suggest a role for Dbr1 interactor AQR in intron recycling. Dbr1 depletion leads to increased dwell time of spliceosome on excised lariats.