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An adapted MS2-MCP system to visualize endogenous cytoplasmic mRNA with live imaging in Caenorhabditis elegans [1]

['Cristina Tocchini', 'Biozentrum', 'University Of Basel', 'Basel', 'Susan E. Mango']

Date: 2024-03

Live imaging of RNA molecules constitutes an invaluable means to track the dynamics of mRNAs, but live imaging in Caenorhabditis elegans has been difficult to achieve. Endogenous transcripts have been observed in nuclei, but endogenous mRNAs have not been detected in the cytoplasm, and functional mRNAs have not been generated. Here, we have adapted live imaging methods to visualize mRNA in embryonic cells. We have tagged endogenous transcripts with MS2 hairpins in the 3′ untranslated region (UTR) and visualized them after adjusting MS2 Coat Protein (MCP) expression. A reduced number of these transcripts accumulates in the cytoplasm, leading to loss-of-function phenotypes. In addition, during epithelial morphogenesis, MS2-tagged mRNAs for dlg-1 fail to associate with the adherens junction, as observed for untagged, endogenous mRNAs. These defects are reversed by inactivating the nonsense-mediated decay pathway. RNA accumulates in the cytoplasm, mutant phenotypes are rescued, and dlg-1 RNA associates with the adherens junction. These data suggest that MS2 repeats can induce the degradation of endogenous RNAs and alter their cytoplasmic distribution. Although our focus is RNAs expressed in epithelial cells during morphogenesis, we find that this method can be applied to other cell types and stages.

Funding: Funding for SEM was provided by the SNSF, grant 310030_197713 and SNF 310030_185157. Funding was also supplied by the Biozentrum, University of Basel to SEM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we establish MS2-MCP tagging to monitor endogenous, functional transcripts for live imaging in C. elegans. The adaptation of the system for C. elegans consists of 3 modifications: (i) a genetic background lacking a proficient nonsense-mediated decay (NMD) pathway to avoid degradation of MS2-tagged transcripts; (ii) fluorescently tagged MCP lacking a nuclear localization signal (NLS) to avoid aberrant sequestration of mRNAs in nuclei; and (iii) a weak promoter to maintain low levels of MCP. These adjustments allow tracking of endogenous transcripts and should prove useful for understanding the dynamics of RNA regulation.

In C. elegans, active sites of transcription have been visualized via a clever genetic trick that tracks fluorescently tagged NRDE-3 in nuclei [ 11 ]. The MS2-MCP system has also been used to visualize nascent RNAs derived from single-copy, integrated transgenes, and this approach has allowed visualization of transcriptional bursting in the nuclei of germline cells [ 12 ]. Overexpressed mRNAs generated from multicopy extrachromosomal arrays have enabled the visualization of RNA dynamics in somatic cells [ 13 ]. Although promising, these examples did not generate functional RNAs from the endogenous locus, raising the question of whether the observed dynamics of transgene RNAs reflected biologically accurate regulation [ 13 ]. The lack of a system to visualize functional, endogenous transcripts in living animals has been a bottleneck to advancing studies of RNA biology in C. elegans.

RNA molecules have a highly dynamic life. Through the different stages of their life, from synthesis to translation, and, ultimately, to degradation, RNAs move between different subcellular compartments and localize to specific subcellular organelles. Tracking an mRNA in real time is pivotal to studying RNA dynamics, which cannot be achieved with fixed samples. Repeat copies of bacteriophage MS2 RNA hairpins have been used extensively to tag RNAs for live imaging [ 1 – 4 ]. The system allows the detection of single mRNA molecules in real time and with high resolution, thanks to high-affinity binding of fluorescently labeled MS2 Coat Protein (MCP) to MS2 hairpins [ 1 ]. This bacteriophage-derived, live-imaging approach has led to a general understanding of the dynamics during the different steps of the complex life of an mRNA, notably transcription, nuclear export, subcellular localization, and degradation [ 1 , 5 – 8 ]. Recent studies have demonstrated the versatility of this system to tackle even more detailed steps of RNA metabolism, such as first round of translation or XRN1-mediatetd 5′-3′ degradation [ 9 , 10 ]. With the advent of the CRISPR/Cas9 technique, it has become possible to genetically engineer MS2 sequences at the endogenous locus. In this way, endogenous, and not reporter, transcripts are detected, allowing a more precise description of what occurs in a cell at the physiological level.

Results

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002526

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