Biology subjects

Aoki, S. T.

Publications and source records attributed to Aoki, S. T..

10 recordsLinked to original sources

bioRxiv2025

eIF4E assembly into C. elegans germ granules is essential for its repressive function

Metazoan germ cells form intracellular germ granules, cytoplasmic RNA-protein condensates that contain a variety of RNAs and proteins essential for germline identity, maintenance, and fertility. P granules are a type of C. elegans germ granule proposed to be sites of mRNA repression. Proper P granule assembly is dependent on PGL-1 and its granule-forming protein relatives. Numerous RNA-binding proteins localize to P granules, like the eIF4E mRNA cap binding homolog, IFE-1. IFE-1 directly interacts with PGL-1 in vivo and in vitro. The molecular function of P granules remains enigmatic. Here, PGL-1 was molecularly dissected in vivo to determine protein regions required for P granule assembly, binding partner recruitment, and germ cell development. A specific region in the PGL-1 C-terminus was necessary and sufficient for IFE-1 recruitment to P granules and for fertility. IFE-1 RNA targets were identified, and reporters of top gene targets were repressed in the adult germline. This repression was dependent on PGL-1 and its IFE-1 binding peptide. These findings provide evidence that IFE-1 and P granules are a factor and site of mRNA repression, respectively. This repression required IFE-1 assembly into P granules, supporting the model that RNA-protein condensate assembly is necessary for its biological and biochemical functions.

developmental biology↗

bioRxiv2025

Exogenous expression of a histone H3.3 isoform causes extranuclear divisions in the C. elegans intestine

Histone proteins condense DNA into chromatin and play significant roles in gene regulation. Mutations that alter histone function can disrupt critical gene expression programs and are implicated in driving cancer and other human diseases. Most mechanistic research on histones has been conducted in cell culture, mitotic tissues, or animal disease models rather than in normal tissue. Caenorhabditis elegans is a model organism with a well-established developmental program and conserved histone mechanisms shared with other metazoans. Prior work generated several single-copy histone reporters expressing exogenous histone protein in the intestine. Surprisingly, the histone H3.3 reporter worms possessed extra nuclei in the anterior intestine. This phenotype was incompletely penetrant and arose early during larval development. Mutations predicted to perturb histone H3.3 reporter expression or deposition into chromatin ameliorated the phenotype. DNA Fluorescence In situ Hybridization (FISH) was used to measure chromosome copy number and revealed approximately half the DNA content in the extra nuclei versus undivided controls, suggesting aberrant nuclear division during normal rounds of intestinal endoreduplication. Thus, these reporters serendipitously identified a distinct function of histone H3.3 in triggering aberrant nuclear division during animal development. Other histone isoforms may have previously unrecognized biological functions in terminally differentiated tissue alongside their roles in mitotic tissue.

developmental biology↗

bioRxiv2025

In vivo pulse-chase in C. elegans reveals intestinal histone turnover changes upon starvation

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 provide 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 C. 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 animals nutritional state. Chromatin Immunoprecipitation and sequencing (ChIP-seq) 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.

biochemistry↗

bioRxiv2024

Accurate in silico predictions of modified RNA interactions to a prototypical RNA-binding protein with {lambda}-dynamics

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 {lambda}-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 {lambda}-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 CHARMM and Amber RNA force fields to determine the best parameter set to use in binding calculations. This work demonstrates that {lambda}-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 {lambda}-dynamics will unlock new frontiers in understanding how RNA modifications shape RNA biochemistry.

biochemistry↗

bioRxiv2024

In silico {lambda}-dynamics predicts protein binding specificities to modified RNAs

RNA modifications shape gene expression through a smorgasbord 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 {lambda}-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. {lambda}-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 {lambda}-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.

biochemistry↗

bioRxiv2024

Effectiveness of a network Open House model to recruit trainees to post-baccalaureate STEM programs

Post-baccalaureate (post-bac) programs can be instrumental in strengthening science training and expanding STEM career opportunities for junior trainees. Many of these sponsored programs are designed to increase research exposure for underrepresented minorities, including African American, Hispanic, Native American, and first-generation college students, among others. Recruiting trainees to post-bac programs can be challenging for reasons including a lack of awareness about available programs. To address this gap, an Open House event was created with the goal of raising awareness and generating interest among potential students for future post-bac programs. Students were recruited from partnering Minority Serving Institutions (MSIs) to attend a two-day event at a Primarily Undergraduate Institution (PUI) and a Research-Intensive (R1) institution. During the visit, students toured both campuses, learned about various post-bac programs and research opportunities, and interacted with faculty, current graduate students, and a former post-bac scholar. Transportation, lodging, and meals were provided. Participants completed voluntary pre- and post-surveys. Results indicated that attendees, the majority of whom were underrepresented minorities in STEM, left with a stronger understanding of post-bac programs and how these experiences could support their future careers in STEM and that students attendance at the event made it more likely they would apply to available post-bac programs. Mentor and MSI faculty survey responses highlighted their strong support for participating in future recruitment events. These findings demonstrate that in-person Open House events, built on collaborative partnerships across institutions, are an effective strategy for increasing awareness and encouraging participation in post-bac training programs-- particularly among underrepresented student populations.

scientific communication and education↗

bioRxiv2023

An effective C. elegans CRISPR training module for high school and undergraduate summer research experiences in molecular biology

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 students curiosity and passion for learning and research. In this study, an effective eight-week training program has been created that encompasses 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, substantial enhancements in students molecular biology proficiency and enthusiasm for scientific exploration was observed. Overall, this diligently crafted training module that employs C. elegans as an educational tool to instruct inexperienced students has demonstrated its accessibility and ability to engage students in molecular biology and gene editing methodologies.

scientific communication and education↗

bioRxiv2021

Tethered NOS-3, a nematode Nanos RNA-binding protein, enhances reporter expression and mRNA stability

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 3UTR harbors functional binding elements for {lambda}N22 peptide, while the mCherry reporter 3UTR 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 N-terminal intrinsically disordered region, not its C-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 RBP tethering to determine its molecular mechanism in a multicellular tissue.

developmental biology↗

bioRxiv2018

Nematode germ granule assembly is linked to mRNA repression

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, called PGL, 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 granules. We determine the crystal structure of the PGL N-terminal region to 1.5 [A], discover its dimerization and identify key residues at the dimer interface. In vivo mutations of those interface residues prevent P-granule assembly, de-repress PGL-tethered mRNA and reduce fertility. Therefore, PGL 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-tethered mRNA. We conclude that P-granule function requires both assembly and localized regulators.

developmental biology↗

bioRxiv2016

An RNA binding polymer specifies nematode sperm fate

Metazoan germ cells develop as sperm or oocytes, depending on chromosomal sex, extrinsic signaling from somatic tissue and intrinsic factors within the germ cells. Gamete fate regulatory networks have been analyzed in nematodes, flies and mammals, but only in C. elegans have terminal intrinsic regulators been identified, which include a Tob/BTG protein family member, FOG-3. Canonical Tob/BTG proteins function as monomeric adaptor proteins that link RNA binding proteins to deadenylases. To ask if FOG-3 functions similarly, we first determined its crystal structure. FOG-3 harbors a classical Tob/BTG fold, but unlike other Tob/BTG proteins, FOG-3 dimerizes and these FOG-3 dimers assemble into polymers. The importance of FOG-3 polymers to sperm fate specification was confirmed in vivo using CRISPR/Cas9 gene editing to create mutations designed to disrupt the polymer interface. The FOG-3 surface potential is highly basic, suggesting binding to nucleic acid. We find that FOG-3 binds RNA directly with a strong preference for 3UTRs of oogenic mRNAs. Our results reveal a divergent but striking molecular assembly for proteins with a Tob/BTG fold, make key advances in understanding the mechanism of sperm fate specification and highlight the potential for undiscovered protein polymers in biology.

developmental biology↗