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Updated: Jul 15, 2025
Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae
Published on: June 14, 2013
Correlative Localization Analysis Between mRNA and Enhanced Green Fluorescence Protein-Fused Protein by a
Yuki Morita1, Yoshinori Katakura1, Kaoru Takegawa1
1Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
Abstract:
Although subcellular localization analysis of proteins fused with enhanced green fluorescence protein (EGFP) has been widely conducted in filamentous fungi, little is known about the localization of messenger RNAs (mRNAs) encoding the EGFP-fused proteins. In this study, we performed single-molecule fluorescence in situ hybridization (smFISH) using an egfp probe to simultaneously visualize EGFP-fused proteins and their mRNAs in the hyphal cells of the filamentous fungus Aspergillus oryzae. We investigated the subcellular localization of mRNAs encoding cytoplasmic EGFP, an actin marker protein Lifeact tagged with EGFP, and several EGFP-fused proteins AoSec22, AoSnc1, AoVam3, and AoUapC that localize to the endoplasmic reticulum (ER), the apical vesicle cluster Spitzenkörper, vacuolar membrane, and plasma membrane, respectively. Visualization of these mRNAs by smFISH demonstrated that each mRNA exhibited distinct localization patterns likely depending on the mRNA sequence. In particular, we revealed that mRNAs encoding Lifeact-EGFP, EGFP-AoSec22, EGFP-AoVam3, and AoUapC-EGFP, but not cytoplasmic EGFP and EGFP-AoSnc1, were preferentially localized at the apical cell, suggesting certain mechanisms to regulate the existence of these transcripts among hyphal regions. Our findings provide the distinct localization information of each mRNA in the hyphal cells of A. oryzae.
Insights
This study visualizes messenger RNAs (mRNAs) for enhanced green fluorescence protein (EGFP)-fused proteins in Aspergillus oryzae. Specific mRNAs, but not all, were found to localize to the hyphal apex, suggesting targeted transcript regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Mycology
Background:
- Subcellular protein localization using enhanced green fluorescence protein (EGFP) fusions is common in filamentous fungi.
- However, the localization of messenger RNAs (mRNAs) encoding these EGFP-fused proteins remains largely uncharacterized.
- Understanding mRNA localization is crucial for deciphering gene expression regulation in fungal hyphae.
Purpose of the Study:
- To investigate the subcellular localization of mRNAs encoding various EGFP-fused proteins in Aspergillus oryzae hyphal cells.
- To determine if mRNA localization patterns differ based on the encoded protein and its known subcellular destination.
- To explore potential mechanisms regulating transcript distribution within fungal hyphae.
Main Methods:
- Employed single-molecule fluorescence in situ hybridization (smFISH) with an EGFP probe.
- Simultaneously visualized EGFP-fused proteins and their corresponding mRNAs.
- Analyzed mRNA localization for cytoplasmic EGFP, Lifeact-EGFP, and EGFP-fused proteins AoSec22, AoSnc1, AoVam3, and AoUapC.
Main Results:
- Each investigated mRNA exhibited distinct subcellular localization patterns within Aspergillus oryzae hyphae.
- mRNAs encoding Lifeact-EGFP, EGFP-AoSec22, EGFP-AoVam3, and AoUapC-EGFP showed preferential localization to the apical cell.
- Cytoplasmic EGFP and EGFP-AoSnc1 mRNAs did not display this apical enrichment, indicating sequence-specific localization.
Conclusions:
- Distinct mRNA localization patterns were observed in Aspergillus oryzae hyphal cells.
- Specific transcripts are preferentially localized to the hyphal apex, suggesting regulated transport or retention mechanisms.
- These findings provide critical insights into mRNA localization and its potential role in spatial gene expression in filamentous fungi.
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