Understanding eGFP-mRNA Role in Protein Localization
We have shown that mRNAs encoding GFP, EBFP2, mCherry, and mOrange associate with the ER. Previously, it was assumed that this association depended on their encoded proteins. Results demonstrated that the zipcode sequence is sufficient to target mRNAs to specific cell locations. Cells expressing the eGFP-mRNA were observed to have translation sites at cell-cell contacts, while those containing the control construct did not.
Targeted to the ER
The fluorescent protein eGFP, originally isolated from the jellyfish Aequorea victoria, is commonly used as a direct detection reporter for mammalian cells. This is due to its ability to provide a strong signal and its wide range of applications, including monitoring cell health and function.
However, only some studies have used the eGFP mRNA technique to investigate the spatial distribution of mRNAs in vivo. Using a Cy5-labeled mRNA encoding, eGFP allowed the visualization of its localization using widefield and confocal fluorescent microscopy. After mRNA in vivo enters the cell, it is targeted to the endoplasmic reticulum (ER) through its interaction with the ER membrane receptor SR.
Once SR binds mRNA, the ribosome-nascent peptide complex is transported to the ER protein production site. The nascent peptide is then bound to the ER translocon, released into the lumen, or incorporated into the ER membrane.
The smFISH method found that mRNA encoding EGFP was localized preferentially to apical regions of A. oryzae hyphae. The mRNA encoding the SNARE protein AoSec22 was evenly distributed among the hyphal regions, and mRNAs encoding the actin proteins were likewise distributed.
The results obtained by smFISH demonstrated that the Cy5-eGFP mRNA was internalized in all types of skin cells that are present in the dermal layers, such as epidermal keratinocytes from the skin surface to 120 mm depth, epithelial cells in appendageal structures, e.g., PSU and sweat glands, vascular tissues, and to a lesser extent dermal fibroblasts. It was also observed that the eGFP expression was not correlated with the injection sites.
Displaced from the ER
A study shows that only a fraction of the eGFP-mRNA localizes to the ER. However, most mRNAs that bind to the IRE1 complex do not exhibit this behavior. Their mobility is more similar to that of secreted Gaussia mRNA, which is generally less mobile than the other WT reporter tracks.
The IRE1-bound mRNAs that do not interact with the ER appear to be targeted to other structures, such as centrosomes or nuclear envelopes, or within cytoplasmic foci that are not P-bodies. These mRNAs do not contain a signal sequence that IRE1 would recognize.
The authors propose that these mRNAs bind to higher-order oligomeric assemblies, which are specialized in targeting specific substrates and may have distinct mechanisms for sorting them to their final destinations. Interestingly, it was shown that depletion of p180 does not affect the percentage of mRNAs associated with the ER, suggesting that other factors influence this association.
Displaced from the Cell Surface
GFP (enhanced green fluorescent protein) is a brightly-colored protein that emits a yellow-green fluorescence at 509 nm and was initially isolated from the jellyfish Aequorea victoria. It is a commonly used reporter for cell-based studies, particularly those focused on protein localization.
GFP has many applications in biology, including labeling living cells for macro-scale observation and photography. GFP is a versatile tool for analyzing cell dynamics and has led to numerous advances in biological research. smFISH is a method that allows for simultaneous co-localization of mRNAs and proteins in the same cells.
This is accomplished by tagging both ends of the mRNA with a marker tag, which can be either a c-terminal eGFP tag or a poly-A tail. The mRNAs are mixed with a housekeeping gene and delivered via nucleofection into cultured cells. After a 24-hour incubation at 37degC, flow cytometry collects and analyzes the cells. smFISH analysis revealed distinct mRNA localization patterns for the indicated genes—for example, mRNAs encoding HMMR, KIF4A, and DYNLL2 accumulated in cytoplasmic protrusions.
In contrast, mRNAs encoding for CRKL and CTNNB1/b-catenin localized to foci. These differences in mRNA localization likely reflect specific protein-based mechanisms. Furthermore, the results indicate that the co-localization of mRNAs with their encoded proteins depends on translational control. Thus, experiments using mRNAs expressing ERN1 target motifs should be carefully interpreted and may be more sensitive to ER stress than those with a silent ERN1 mutant mRNA.
Displaced from the Hyphal Tip
Using smFISH, we confirmed the localization of eGFP-mRNA to hyphal tips and found that eGFP mRNAs are not randomly distributed. Moreover, we observed a clear correlation between the mRNA distribution and gene annotation. This suggests that mRNAs are transported to the hyphal tip region and translated into proteins after their nucleus has received appropriate environmental cues.
The mRNA distribution in the hyphal cell can be divided into the apical, middle, and basal regions. smFISH was used with HeLa cells expressing BAC-tagged mRNAs encoding for the SNARE proteins ER and Spitzenkorper to test the hypothesis that mRNAs are transported to the tip.
It was found that mRNAs encoding ER-localized proteins are preferentially localized to apical regions, while those encoding Spitzenkorper are evenly distributed in hyphal extensions. These results suggest that mRNAs encoding for SNARE proteins need to exist at the apical region of the hypha to initiate the secretory pathway.
