Abstract

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.

Details

Title
The debranching enzyme Dbr1 regulates lariat turnover and intron splicing
Author
Buerer, Luke 1   VIAFID ORCID Logo  ; Clark, Nathaniel E. 1   VIAFID ORCID Logo  ; Welch, Anastasia 1 ; Duan, Chaorui 1 ; Taggart, Allison J. 1 ; Townley, Brittany A. 2 ; Wang, Jing 1 ; Soemedi, Rachel 1 ; Rong, Stephen 3   VIAFID ORCID Logo  ; Lin, Chien-Ling 4   VIAFID ORCID Logo  ; Zeng, Yi 5 ; Katolik, Adam 6 ; Staley, Jonathan P. 7   VIAFID ORCID Logo  ; Damha, Masad J. 6   VIAFID ORCID Logo  ; Mosammaparast, Nima 2   VIAFID ORCID Logo  ; Fairbrother, William G. 8 

 Brown University, Department of Molecular Biology, Cell Biology, and Biochemistry, Providence, USA (GRID:grid.40263.33) (ISNI:0000 0004 1936 9094) 
 Washington University School of Medicine, Department of Pathology & Immunology, Center for Genome Integrity, St. Louis, USA (GRID:grid.4367.6) (ISNI:0000 0001 2355 7002) 
 Brown University, Center for Computational Molecular Biology, Providence, USA (GRID:grid.40263.33) (ISNI:0000 0004 1936 9094); Yale University, Department of Genetics, New Haven, USA (GRID:grid.47100.32) (ISNI:0000 0004 1936 8710) 
 Brown University, Department of Molecular Biology, Cell Biology, and Biochemistry, Providence, USA (GRID:grid.40263.33) (ISNI:0000 0004 1936 9094); Academia Sinica, Institute of Molecular Biology, Taipei, Taiwan (GRID:grid.28665.3f) (ISNI:0000 0001 2287 1366) 
 University of Chicago, Department of Molecular Genetics and Cell Biology, Chicago, USA (GRID:grid.170205.1) (ISNI:0000 0004 1936 7822); Stanford University School of Medicine, Department of Genetics, Stanford, USA (GRID:grid.168010.e) (ISNI:0000000419368956) 
 McGill University, Department of Chemistry, Montreal, Canada (GRID:grid.14709.3b) (ISNI:0000 0004 1936 8649) 
 University of Chicago, Department of Molecular Genetics and Cell Biology, Chicago, USA (GRID:grid.170205.1) (ISNI:0000 0004 1936 7822) 
 Brown University, Department of Molecular Biology, Cell Biology, and Biochemistry, Providence, USA (GRID:grid.40263.33) (ISNI:0000 0004 1936 9094); Brown University, Center for Computational Molecular Biology, Providence, USA (GRID:grid.40263.33) (ISNI:0000 0004 1936 9094) 
Pages
4617
Publication year
2024
Publication date
2024
Publisher
Nature Publishing Group
e-ISSN
20411723
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
3062308474
Copyright
© The Author(s) 2024. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.