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Single-molecule imaging reveals switch between spurious and functional ncRNA transcription

Wednesday, September 16, 2015 — Poster Session I

3:30 p.m. – 5:00 p.m.
FAES Terrace
NCI
MOLBIO-22

* FARE Award Winner

Authors

  • TL Lenstra
  • A Coulon
  • C Chow
  • DR Larson

Abstract

Genomic data indicates that eukaryotic genomes are ubiquitously transcribed, but the function of non-coding RNAs is largely unknown. In this study, we use time-lapse single-molecule imaging in living cells in tandem with strand-specific transcriptional blocking of the ncRNA by CRISPR/Cas9d to interrogate the role of antisense ncRNA transcription at the GAL locus of S. cerevisiae. Both coding and non-coding RNA syntheses were visualized in single cells in real-time using the PP7 and MS2 RNA labeling technique. We observed ncRNA synthesis before, and even during sense transcription, suggesting that the template is highly permissive for transcription. By targeting an enzymatically-dead mutant of Cas9, we were able to selectively block transcription of one strand without affecting transcription of the opposite strand. Blocking ncRNA in this manner under repressive conditions results in increased transcriptional leakage of two genes at the locus, one of which encodes a signaling molecule. Leakage of this signaling molecule alters the activation threshold, and hysteresis of the network. However, blocking ncRNA had no detectable effects on the actively transcribed gene. Using a computational model we were able to quantitatively determine that the ncRNA controls the initial conditions of signaling molecules, but does not alter the topology or rate constants of the network. We thus experimentally uncouple the appearance of ncRNA from the function of the ncRNA. In conclusion, we find that transcription of the same ncRNA is functional under repressive conditions but spurious and pervasive under activating conditions, highlighting the nuanced roles that ncRNA can play in gene regulation

Category: Molecular Biology and Biochemistry