title: Stem-loop RNA labeling can affect nuclear and cytoplasmic mRNA processing.
abstract: The binding of sequence-specific RNA-interacting proteins, such as the bacteriophage MS2 or PP7 coat proteins, to their corresponding target sequences has been extremely useful and widely used to visualize single mRNAs in vivo. However, introduction of MS2 stem-loops into yeast mRNAs has recently been shown to lead to the accumulation of RNA fragments, suggesting that the loops impair mRNA decay. This result was questioned, because fragment occurrence was mainly assessed using ensemble methods, and their cellular localization and its implications had not been addressed on a single transcript level. Here, we demonstrate that the introduction of either MS2 stem-loops (MS2SL) or PP7 stem-loops (PP7SL) can affect the processing and subcellular localization of mRNA. We use single-molecule fluorescence in situ hybridization (smFISH) to determine the localization of three independent mRNAs tagged with the stem-loop labeling systems in glucose-rich and glucose starvation conditions. Transcripts containing MS2SL or PP7SL display aberrant localization in both the nucleus and the cytoplasm. These defects are most prominent in glucose starvation conditions, with nuclear mRNA processing being altered and stem-loop fragments abnormally enriching in processing bodies (PBs). The mislocalization of SL-containing RNAs is independent of the presence of the MS2 or PP7 coat protein (MCP or PCP).
authors.affiliation: Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Heinrich
authors.affiliation: Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland.
authors.forename: Corinne L
authors.initials: CL
authors.lastname: Sidler
authors.affiliation: Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland.
authors.forename: Claus M
authors.initials: CM
authors.lastname: Azzalin
authors.affiliation: Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland.
authors.forename: Karsten
authors.identifier: 0000-0001-7224-925X
authors.initials: K
authors.lastname: Weis
mesh_terms: D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D017404:In Situ Hybridization, Fluorescence; D055029:Inverted Repeat Sequences; D017909:Levivirus / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D020871:RNA Stability; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D000072760:Single Molecule Imaging; D013194:Staining and Labeling / Q000379:methods; D014162:Transfection
chemical_list: D036022:Capsid Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D005947:Glucose
keywords: MS2–MCP system; P bodies; PP7–PCP system; mRNA decay; mRNA processing; single-molecule fluorescence in situ hybridization