Cytoplasmic RNA–protein (RNP) granules have diverse biophysical properties, from liquid to solid, and play enigmatic roles in RNA metabolism. Nematode P granules are paradigmatic liquid droplet granules and central to germ cell development. Here we analyze a key P granule scaffolding protein, PGL-1, to investigate the functional relationship between P granule assembly and function. Using a protein–RNA tethering assay, we find that reporter mRNA expression is repressed when recruited to PGL-1. We determine the crystal structure of the PGL-1 N-terminal region to 1.5 Å, discover its dimerization, and identify key residues at the dimer interface. Mutations of those interface residues prevent P granule assembly in vivo, de-repress PGL-1 tethered mRNA, and reduce fertility. Therefore, PGL-1 dimerization lies at the heart of both P granule assembly and function. Finally, we identify the P granule-associated Argonaute WAGO-1 as crucial for repression of PGL-1 tethered mRNA. We conclude that P granule function requires both assembly and localized regulators.

The ability to study protein dynamics and function in the authentic context of a multicellular organism is paramount to better understand biological phenomena in animal health and disease. Pulse-chase of self-labeling fusion protein tags provides the opportunity to label proteins of interest and track those proteins over time. There are currently several challenges associated with performing in vivo protein pulse-chase in animals, such as cost, reproducibility, and accurate detection methods. The Caenorhabditis elegans model organism has attributes that alleviate many of these challenges. This work tests the feasibility of applying the Halo modified enzyme (HaloTag) for in vivo protein pulse-chase in C. elegans. HaloTag intestinal histone reporters were created in the worm and used to demonstrate that reporter protein could be efficiently pulse-labeled by soaking animals in ligand. Labeled protein stability could be monitored over time by fluorescent confocal microscopy. Further investigation revealed reporter protein stability was dependent on the animal's nutritional state. Chromatin immunoprecipitation and sequencing of the reporters showed incorporation in chromatin with little change hours into starvation, implying a lack of chromatin regulation at the time point tested. Collectively, this work presents a straightforward method to label and track proteins of interest in C. elegans that can address a multitude of biological questions surrounding protein stability and dynamics in this animal model.

Engaging in research experiences as a high school or undergraduate student interested in science, technology, engineering, and mathematics (STEM) is pivotal for their academic and professional development. A structured teaching framework can help cultivate a student's curiosity and passion for learning and research. In this study, an eight‐week training program was created to encompass fundamental molecular biology principles and hands‐on laboratory activities. This curriculum focuses on using clustered regularly interspaced short palindromic repeats (CRISPR) gene editing in the Caenorhabditis elegans model organism. Through pre‐ and post‐program assessments, enhancements in students' molecular biology proficiency and enthusiasm for scientific exploration were observed. Overall, this training module demonstrated its accessibility and ability to engage inexperienced students in molecular biology and gene editing methodologies.

Robust methods are critical for testing the in vivo regulatory mechanism of RNA binding proteins. Here we report improvement of a protein–mRNA tethering assay to probe the function of an RNA binding protein in its natural context within the C. elegans adult germline. The assay relies on a dual reporter expressing two mRNAs from a single promoter and resolved by trans-splicing. The gfp reporter 3′UTR harbors functional binding elements for λN22 peptide, while the mCherry reporter 3′UTR carries mutated nonfunctional elements. This strategy enables internally controlled quantitation of reporter protein by immunofluorescence and mRNA by smFISH. To test the new system, we analyzed a C. elegans Nanos protein, NOS-3, which serves as a post-transcriptional regulator of germ cell fate. Unexpectedly, tethered NOS-3 enhanced reporter expression. We confirmed this enhancement activity with a second reporter engineered at an endogenous germline gene. NOS-3 enhancement of reporter expression was associated with its amino-terminal intrinsically disordered region, not its carboxy-terminal zinc fingers. RNA quantitation revealed that tethered NOS-3 enhances stability of the reporter mRNA. We suggest that this direct NOS-3 enhancement activity may explain a paradox: Classically Nanos proteins are expected to repress RNA, but nos-3 had been found to promote gld-1 expression, an effect that could be direct. Regardless, the new dual reporter dramatically improves in situ quantitation of reporter expression after RNA binding protein tethering to determine its molecular mechanism in a multicellular tissue.

RNA modifications shape gene expression through a variety of chemical changes to canonical RNA bases. Although numbering in the hundreds, only a few RNA modifications are well characterized, in part due to the absence of methods to identify modification sites. Antibodies remain a common tool to identify modified RNA and infer modification sites through straightforward applications. However, specificity issues can result in off-target binding and confound conclusions. This work utilizes in silico λ-dynamics to efficiently estimate binding free energy differences of modification-targeting antibodies between a variety of naturally occurring RNA modifications. Crystal structures of inosine and N6-methyladenosine (m6A) targeting antibodies bound to their modified ribonucleosides were determined and served as structural starting points. λ-Dynamics was utilized to predict RNA modifications that permit or inhibit binding to these antibodies. In vitro RNA-antibody binding assays supported the accuracy of these in silico results. High agreement between experimental and computed binding propensities demonstrated that λ-dynamics can serve as a predictive screen for antibody specificity against libraries of RNA modifications. More importantly, this strategy is an innovative way to elucidate how hundreds of known RNA modifications interact with biological molecules without the limitations imposed by in vitro or in vivo methodologies.

RNA-binding proteins shape biology through their widespread functions in RNA biochemistry. Their function requires the recognition of specific RNA motifs for targeted binding. These RNA binding elements can be composed of both unmodified and chemically modified RNAs, of which over 170 chemical modifications have been identified in biology. Unmodified RNA sequence preferences for RNA-binding proteins have been widely studied, with numerous methods available to identify their preferred sequence motifs. However, only a few techniques can detect preferred RNA modifications, and no current method can comprehensively screen the vast array of hundreds of natural RNA modifications. Prior work demonstrated that λ-dynamics is an accurate in silico method to predict RNA base binding preferences of an RNA-binding antibody. This work extends that effort by using λ-dynamics to predict unmodified and modified RNA binding preferences of human Pumilio, a prototypical RNA-binding protein. A library of RNA modifications was screened at eight nucleotide positions along the RNA to identify modifications predicted to affect Pumilio binding. Computed binding affinities were compared with experimental data to reveal high predictive accuracy. In silico force field accuracies were also evaluated between CHARMM36 and Amber RNA force fields to determine the best parameter set to use in binding calculations. This work demonstrates that λ-dynamics can predict RNA interactions to a bona fide RNA-binding protein without the requirements of chemical reagents or new methods to experimentally test binding at the bench. Advancing in silico methods like λ-dynamics will unlock new frontiers in understanding how RNA modifications shape RNA biochemistry.