Kinetic competition during the transcription cycle results in stochastic RNA processing

Elife. 2014 Oct 1;3:e03939. doi: 10.7554/eLife.03939.

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.

Keywords: RNA processing; biophysics; chromosomes; fluctuation analysis; genes; human; single-molecule imaging; splicing; structural biology; transcription.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Line, Tumor
  • Cell Survival
  • Chromatin / metabolism
  • Computer Systems
  • Diffusion
  • Humans
  • Kinetics
  • Mutant Proteins / metabolism
  • Mutation / genetics
  • Neoplasms / genetics
  • Nuclear Proteins / metabolism
  • RNA / genetics*
  • RNA Processing, Post-Transcriptional / genetics*
  • RNA Splicing / genetics
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Ribonucleoproteins / metabolism
  • Splicing Factor U2AF
  • Stochastic Processes
  • Transcription, Genetic*
  • beta-Globins / genetics
  • beta-Globins / metabolism

Substances

  • Chromatin
  • Mutant Proteins
  • Nuclear Proteins
  • RNA, Messenger
  • Ribonucleoproteins
  • Splicing Factor U2AF
  • U2AF1 protein, human
  • beta-Globins
  • RNA