An improved MS2 system for accurate reporting of the mRNA life cycle

Evelina Tutucci, Maria Vera Ugalde, Jeetayu Biswas, Jennifer Garcia, Roy Parker, Robert H. Singer

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

The MS2-MCP system enables researchers to image multiple steps of the mRNA life cycle with high temporal and spatial resolution. However, for short-lived mRNAs, the tight binding of the MS2 coat protein (MCP) to the MS2 binding sites (MBS) protects the RNA from being efficiently degraded, and this confounds the study of mRNA regulation. Here, we describe a reporter system (MBSV6) with reduced affinity for the MCP, which allows mRNA degradation while preserving single-molecule detection determined by single-molecule FISH (smFISH) or live imaging. Constitutive mRNAs (MDN1 and DOA1) and highly-regulated mRNAs (GAL1 and ASH1) endogenously tagged with MBSV6 in Saccharomyces cerevisiae degrade normally. As a result, short-lived mRNAs were imaged throughout their complete life cycle. The MBSV6 reporter revealed that, in contrast to previous findings, coordinated recruitment of mRNAs at specialized structures such as P-bodies during stress did not occur, and mRNA degradation was heterogeneously distributed in the cytoplasm.

Original languageEnglish (US)
Pages (from-to)81-89
Number of pages9
JournalNature Methods
Volume15
Issue number1
DOIs
StatePublished - Jan 3 2018

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Life Cycle Stages
Life cycle
Messenger RNA
Capsid Proteins
RNA Stability
Degradation
Molecules
Saccharomyces cerevisiae
Cytoplasm
Binding Sites
Research Personnel
Yeast
RNA
Imaging techniques

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Tutucci, E., Vera Ugalde, M., Biswas, J., Garcia, J., Parker, R., & Singer, R. H. (2018). An improved MS2 system for accurate reporting of the mRNA life cycle. Nature Methods, 15(1), 81-89. https://doi.org/10.1038/nmeth.4502

An improved MS2 system for accurate reporting of the mRNA life cycle. / Tutucci, Evelina; Vera Ugalde, Maria; Biswas, Jeetayu; Garcia, Jennifer; Parker, Roy; Singer, Robert H.

In: Nature Methods, Vol. 15, No. 1, 03.01.2018, p. 81-89.

Research output: Contribution to journalArticle

Tutucci, E, Vera Ugalde, M, Biswas, J, Garcia, J, Parker, R & Singer, RH 2018, 'An improved MS2 system for accurate reporting of the mRNA life cycle', Nature Methods, vol. 15, no. 1, pp. 81-89. https://doi.org/10.1038/nmeth.4502
Tutucci, Evelina ; Vera Ugalde, Maria ; Biswas, Jeetayu ; Garcia, Jennifer ; Parker, Roy ; Singer, Robert H. / An improved MS2 system for accurate reporting of the mRNA life cycle. In: Nature Methods. 2018 ; Vol. 15, No. 1. pp. 81-89.
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