The ERM-1 membrane-binding domain directs erm-1 mRNA localization to the plasma membrane in the C. elegans embryo.

mRNA localization and transport are integral in regulating gene expression. In Caenorhabditis elegans embryos, the maternally inherited mRNA erm-1 (Ezrin/Radixin/Moesin) becomes concentrated in anterior blastomeres. erm-1 mRNA localizes within those blastomeres to the plasma membrane where the essential ERM-1 protein, a membrane-actin linker, is also found. We demonstrate that the localization of erm-1 mRNA to the plasma membrane is translation dependent and requires its encoded N-terminal, membrane-binding (FERM) domain. By perturbing translation through multiple methods, we found that erm-1 mRNA localization at the plasma membrane persisted only if the nascent peptide remained in complex with the translating mRNA. Indeed, re-coding the erm-1 mRNA coding sequence while preserving the encoded amino acid sequence did not disrupt erm-1 mRNA localization, corroborating that the information directing mRNA localization resides within its membrane-binding protein domain. A single-molecule inexpensive fluorescence in situ hybridization screen of 17 genes encoding similar membrane-binding domains identified three plasma membrane-localized mRNAs in the early embryo. Ten additional transcripts showed potential membrane localization later in development. These findings point to a translation-dependent pathway for localization of mRNAs encoding membrane-associated proteins.

It's Just a Phase: Exploring the Relationship Between mRNA, Biomolecular Condensates, and Translational Control.

Cells spatially organize their molecular components to carry out fundamental biological processes and guide proper development. The spatial organization of RNA within the cell can both promote and result from gene expression regulatory control. Recent studies have demonstrated diverse associations between RNA spatial patterning and translation regulatory control. One form of patterning, compartmentalization in biomolecular condensates, has been of particular interest. Generally, transcripts associated with cytoplasmic biomolecular condensates-such as germ granules, stress granules, and P-bodies-are linked with low translational status. However, recent studies have identified new biomolecular condensates with diverse roles associated with active translation. This review outlines RNA compartmentalization in various condensates that occur in association with repressed or active translational states, highlights recent findings in well-studied condensates, and explores novel condensate behaviors.

Improved Methods for Single-Molecule Fluorescence In Situ Hybridization and Immunofluorescence in Caenorhabditis elegans Embryos.

Visualization of gene products in Caenorhabditis elegans has provided insights into the molecular and biological functions of many novel genes in their native contexts. Single-molecule fluorescence in situ hybridization (smFISH) and immunofluorescence (IF) enable the visualization of the abundance and localization of mRNAs and proteins, respectively, allowing researchers to ultimately elucidate the localization, dynamics, and functions of the corresponding genes. Whereas both smFISH and immunofluorescence have been foundational techniques in molecular biology, each protocol poses challenges for use in the C. elegans embryo. smFISH protocols suffer from high initial costs and can photobleach rapidly, and immunofluorescence requires technically challenging permeabilization steps and slide preparation. Most importantly, published smFISH and IF protocols have predominantly been mutually exclusive, preventing the exploration of relationships between an mRNA and a relevant protein in the same sample. Here, we describe protocols to perform immunofluorescence and smFISH in C. elegans embryos either in sequence or simultaneously. We also outline the steps to perform smFISH or immunofluorescence alone, including several improvements and optimizations to existing approaches. These protocols feature improved fixation and permeabilization steps to preserve cellular morphology while maintaining probe and antibody accessibility in the embryo, a streamlined, in-tube approach for antibody staining that negates freeze-cracking, a validated method to perform the cost-reducing single molecule inexpensive FISH (smiFISH) adaptation, slide preparation using empirically determined optimal antifade products, and straightforward quantification and data analysis methods. Finally, we discuss tricks and tips to help the reader optimize and troubleshoot individual steps in each protocol. Together, these protocols simplify existing workflows for single-molecule RNA and protein detection. Moreover, simultaneous, high-resolution imaging of proteins and RNAs of interest will permit analysis, quantification, and comparison of protein and RNA distributions, furthering our understanding of the relationship between RNAs and their protein products or cellular markers in early development. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sequential immunofluorescence and single-molecule fluorescence in situ hybridization Alternate Protocol: Abbreviated protocol for simultaneous immunofluorescence and single-molecule fluorescence in situ hybridization Basic Protocol 2: Simplified immunofluorescence in C. elegans embryos Basic Protocol 3: Single-molecule fluorescence in situ hybridization or single-molecule inexpensive fluorescence in situ hybridization.

mRNA localization is linked to translation regulation in the Caenorhabditis elegans germ lineage.

Caenorhabditis elegans early embryos generate cell-specific transcriptomes despite lacking active transcription, thereby presenting an opportunity to study mechanisms of post-transcriptional regulatory control. We observed that some cell-specific mRNAs accumulate non-homogenously within cells, localizing to membranes, P granules (associated with progenitor germ cells in the P lineage) and P-bodies (associated with RNA processing). The subcellular distribution of transcripts differed in their dependence on 3'UTRs and RNA binding proteins, suggesting diverse regulatory mechanisms. Notably, we found strong but imperfect correlations between low translational status and P granule localization within the progenitor germ lineage. By uncoupling translation from mRNA localization, we untangled a long-standing question: Are mRNAs directed to P granules to be translationally repressed, or do they accumulate there as a consequence of this repression? We found that translational repression preceded P granule localization and could occur independently of it. Further, disruption of translation was sufficient to send homogenously distributed mRNAs to P granules. These results implicate transcriptional repression as a means to deliver essential maternal transcripts to the progenitor germ lineage for later translation.

Todos Santos small RNA symposium.

Worm biologists from the United States, Canada, and the United Kingdom gathered at the Colorado State University Todos Santos Center in Baja California Sur, Mexico, April 3-5, 2019 for the Todos Santos Small RNA Symposium. Meeting participants, many of whom were still recovering from the bomb cyclone that struck a large swath of North America just days earlier, were greeted by the warmth and sunshine that is nearly ubiquitous in the sleepy seaside town of Todos Santos. With only 24 speakers, the meeting had the sort of laid-back vibe you might expect amongst the palm trees and ocean breeze of the Pacific coast of Mexico. The meeting started with tracing the laboratory lineages of participants. Not surprisingly, the most common parental lineages represented at the meeting were Dr. Craig Mello, Dr. Gary Ruvkun, and Dr. Victor Ambros, whom, together with Dr. Andy Fire and Dr. David Baulcombe, pioneered the small RNA field. In sad irony, on the closing day of the meeting, participants were met with the news of Dr. Sydney Brenner's passing. By establishing the worm, Caenorhabditis elegans, as a model system Dr. Brenner paved the way for much of the research discussed here.