title: Kinetic competition during the transcription cycle results in stochastic RNA processing.
abstract: Synthesis of mRNA in eukaryotes involves the coordinated action of many enzymatic processes, including initiation, elongation, splicing, and cleavage. Kinetic competition between these processes has been proposed to determine RNA fate, yet such coupling has never been observed in vivo on single transcripts. In this study, we use dual-color single-molecule RNA imaging in living human cells to construct a complete kinetic profile of transcription and splicing of the β-globin gene. We find that kinetic competition results in multiple competing pathways for pre-mRNA splicing. Splicing of the terminal intron occurs stochastically both before and after transcript release, indicating there is not a strict quality control checkpoint. The majority of pre-mRNAs are spliced after release, while diffusing away from the site of transcription. A single missense point mutation (S34F) in the essential splicing factor U2AF1 which occurs in human cancers perturbs this kinetic balance and defers splicing to occur entirely post-release.
authors.affiliation: Laboratory of Biological Modeling, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, United States.
authors.forename: Antoine
authors.initials: A
authors.lastname: Coulon
authors.affiliation: Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, United States.
authors.forename: Matthew L
authors.identifier: http://orcid.org/0000-0003-0760-757X
authors.initials: ML
authors.lastname: Ferguson
authors.affiliation: Center for Life Nanoscience, Istituto Italiano di Tecnologia, Rome, Italy.
authors.forename: Valeria
authors.identifier: http://orcid.org/0000-0003-0872-185X
authors.initials: V
authors.lastname: de Turris
authors.affiliation: Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, United States.
authors.forename: Murali
authors.initials: M
authors.lastname: Palangat
authors.affiliation: Laboratory of Biological Modeling, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, United States.
authors.forename: Carson C
authors.initials: CC
authors.lastname: Chow
authors.affiliation: Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, United States.
authors.forename: Daniel R
authors.identifier: http://orcid.org/0000-0001-9253-3055
authors.initials: DR
authors.lastname: Larson
mesh_terms: D045744:Cell Line, Tumor; D002470:Cell Survival; D002843:Chromatin / Q000378:metabolism; D003199:Computer Systems; D004058:Diffusion; D006801:Humans; D007700:Kinetics; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009369:Neoplasms / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D012313:RNA / Q000235:genetics*; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D000072668:Splicing Factor U2AF; D013269:Stochastic Processes; D014158:Transcription, Genetic; D055544:beta-Globins / Q000235:genetics / Q000378:metabolism
chemical_list: D002843:Chromatin; D050505:Mutant Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C076753:U2AF1 protein, human; D055544:beta-Globins; D012313:RNA
keywords: RNA processing; biophysics; chromosomes; fluctuation analysis; genes; human; single-molecule imaging; splicing; structural biology; transcription