Imaging of endogenous messenger RNA splice variants in living cells reveals nuclear retention of transcripts inaccessible to nonsense-mediated decay in Arabidopsis

Janett Göhring, Jaroslaw Jacak, Andrea Barta

Research output: Contribution to journalArticlepeer-review

62 Citations (Scopus)

Abstract

Alternative splicing (AS) is an important regulatory process that leads to the creation of multiple RNA transcripts from a single gene. Alternative transcripts often carry premature termination codons (PTCs), which trigger nonsense-mediated decay (NMD), a cytoplasmic RNA degradation pathway. However, intron retention, the most prevalent AS event in plants, often leads to PTC-carrying splice variants that are insensitive to NMD; this led us to question the fate of these special RNA variants. Here, we present an innovative approach to monitor and characterize endogenous mRNA splice variants within living plant cells. This method combines standard confocal laser scanning microscopy for molecular beacon detection with a robust statistical pipeline for sample comparison. We demonstrate this technique on the localization of NMD-insensitive splice variants of two Arabidopsis thaliana genes, RS2Z33 and the SEF factor. The experiments reveal that these intron-containing splice variants remain within the nucleus, which allows them to escape the NMD machinery. Moreover, fluorescence recovery after photobleaching experiments in the nucleoplasm show a decreased mobility of intron-retained mRNAs compared with fully spliced RNAs. In addition, differences in mobility were observed for an mRNA dependent on its origin from an intron-free or an intron-containing gene.

Original languageEnglish
Pages (from-to)754-764
Number of pages11
JournalThe Plant Cell
Volume26
Issue number2
DOIs
Publication statusPublished - Feb 2014

Keywords

  • Alternative Splicing/genetics
  • Arabidopsis/cytology
  • Arabidopsis Proteins/genetics
  • Cell Nucleus/metabolism
  • Cell Survival
  • Electroporation
  • Fluorescence Recovery After Photobleaching
  • Molecular Imaging/methods
  • Nonsense Mediated mRNA Decay/genetics
  • Plant Cells/metabolism
  • Protoplasts/cytology
  • RNA, Messenger/genetics
  • Transfection

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