TY - JOUR
T1 - Imaging of endogenous messenger RNA splice variants in living cells reveals nuclear retention of transcripts inaccessible to nonsense-mediated decay in Arabidopsis
AU - Göhring, Janett
AU - Jacak, Jaroslaw
AU - Barta, Andrea
N1 - Copyright:
Copyright 2014 Elsevier B.V., All rights reserved.
PY - 2014/2
Y1 - 2014/2
N2 - 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.
AB - 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.
KW - Alternative Splicing/genetics
KW - Arabidopsis/cytology
KW - Arabidopsis Proteins/genetics
KW - Cell Nucleus/metabolism
KW - Cell Survival
KW - Electroporation
KW - Fluorescence Recovery After Photobleaching
KW - Molecular Imaging/methods
KW - Nonsense Mediated mRNA Decay/genetics
KW - Plant Cells/metabolism
KW - Protoplasts/cytology
KW - RNA, Messenger/genetics
KW - Transfection
UR - http://www.scopus.com/inward/record.url?scp=84897039206&partnerID=8YFLogxK
U2 - 10.1105/tpc.113.118075
DO - 10.1105/tpc.113.118075
M3 - Article
C2 - 24532591
SN - 1532-298X
VL - 26
SP - 754
EP - 764
JO - The Plant Cell
JF - The Plant Cell
IS - 2
ER -