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Stem-loop RNA labeling can affect nuclear and cytoplasmic mRNA processing

Stephanie Heinrich1, Corinne L Sidler1, Claus M Azzalin1

  • 1Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland.

RNA (New York, N.Y.)
|January 19, 2017
PubMed

Insights

Introducing MS2 or PP7 stem-loops into yeast mRNA can disrupt RNA processing and localization, particularly under starvation conditions. These stem-loops cause aberrant mRNA distribution and fragment accumulation, independent of coat proteins.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • RNA Biology

Background:

  • Sequence-specific RNA-binding proteins like MS2 and PP7 coat proteins are used for visualizing single mRNAs in vivo.
  • Previous studies suggested MS2 stem-loops impair yeast mRNA decay, but this was based on ensemble methods and lacked single-transcript localization data.

Purpose of the Study:

  • To investigate the impact of MS2 stem-loops (MS2SL) and PP7 stem-loops (PP7SL) on mRNA processing and subcellular localization at the single-molecule level.
  • To determine if stem-loop introduction affects mRNA localization under varying nutrient conditions.

Main Methods:

  • Single-molecule fluorescence in situ hybridization (smFISH) was employed to track the localization of three distinct mRNAs tagged with MS2SL or PP7SL.
  • Experiments were conducted in yeast cells under both glucose-rich and glucose starvation conditions.

Main Results:

  • Introduction of MS2SL or PP7SL leads to aberrant nuclear and cytoplasmic localization of tagged mRNAs.
  • These localization defects are exacerbated during glucose starvation, affecting nuclear mRNA processing.
  • Stem-loop fragments were observed to abnormally accumulate in processing bodies (PBs).
  • Mislocalization was independent of the MS2 or PP7 coat proteins (MCP or PCP).

Conclusions:

  • MS2 and PP7 stem-loops can significantly alter mRNA processing and subcellular localization in yeast.
  • Nutrient stress, such as glucose starvation, amplifies these stem-loop-induced defects.
  • The observed mislocalization is an intrinsic property of the stem-loops themselves, not dependent on their bound proteins.
Keywords:
MS2–MCP systemP bodiesPP7–PCP systemmRNA decaymRNA processingsingle-molecule fluorescence in situ hybridization

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