Biology subjects

Hundley, H. A.

Publications and source records attributed to Hundley, H. A..

8 recordsLinked to original sources

bioRxiv2025

ADARs mediate distinct RNA editing activity and gene regulation in the Caenorhabditis elegans germline

Tissues rely on unique landscapes of gene regulation to allow the organism to correctly develop, function, and respond to changes. One component of these gene regulatory networks is RNA Binding Proteins (RBPs) which bind and modify RNA molecules leading to changes in the cellular fate of transcripts. The Adenosine DeAminase acting on RNA (ADAR) family of RBPs modify RNAs by catalyzing the deamination of adenosine (A) to inosine (I), known as A-to-I RNA editing. Prompted by recent evidence that ADARs play important roles in germline biology, we profiled editing activity of the A-to-I editing enzyme ADR-2 on transcripts in the Caenorhabditis elegans germline. These analyses revealed that many germline editing events are distinct from editing events in other tissues; however, the previously described role of the inactive deaminase ADR-1 in regulating editing activity by ADR-2 is conserved in the germline. We find that complete loss or misregulation of editing has little effect on the expression of edited transcripts within the germline; however, loss of ADARs results in the misexpression of several unedited germline transcripts. Intriguingly, further investigation reveals that these expression changes are buffered at the translational level. In all, the results of this study suggest that ADARs show unique activity in the C. elegans germline and that compensatory mechanisms exist to lessen the immediate consequences of loss of ADAR function within the germline.

molecular biology↗

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

RNA binding by ADARs prevents RNA interference from attacking self-produced dsRNA

The ability of an organism to identify self and foreign RNA is central to eliciting an immune response in times of need while avoiding autoimmunity. As viral pathogens typically employ double-stranded RNA (dsRNA), host identification, modulation, and response to dsRNA is key. However, dsRNA is also abundant in host transcriptomes, raising the question of how these molecules can be differentiated. Two host pathways that regulate dsRNA are A-to-I RNA editing by adenosine deaminases (ADARs), and RNA interference (RNAi). Both mechanisms are important for normal organism development and function by regulating gene expression. Herein, we studied the structure and amount of siRNAs at editing sites and the ability of ADARs to prevent exogenous RNAi using the model organism, Caenorhabditis elegans. We found that the number of siRNAs targeting edited genes is significantly upregulated in ADAR mutant animals. We also found that despite an almost complete depletion of primary siRNAs generated from editing sites in wildtype animals, secondary siRNAs are generated from edited transcripts, suggesting ADARs antagonize only the first step of RNAi processing. We show that ADARs interfere with the efficacy of exogenous RNAi in vivo, probably to prevent trans-silencing, and have indications that ADR-2 binding to the dsRNA is needed for the efficient prevention of RNAi. This work sheds light on how the RNA editing process protects self-produced dsRNAs from aberrant recognition by the immune processes in the cell and from by-product degradation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/672520v2_ufig1.gif" ALT="Figure 1"> View larger version (72K): org.highwire.dtl.DTLVardef@b34f2corg.highwire.dtl.DTLVardef@130c88org.highwire.dtl.DTLVardef@9be2eborg.highwire.dtl.DTLVardef@4ebeec_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

A high-throughput screen identifies RNA binding proteins that affect fertility in Caenorhabditis elegans and reveals a functional relationship between ADR-2 and SQD-1

RNA binding proteins play essential roles in coordinating germline gene expression and development in all organisms. Here, we report that loss of ADR-2, a member of the Adenosine DeAminase acting on RNA (ADAR) family of RNA binding proteins and the sole adenosine-to-inosine RNA editing enzyme in C. elegans, can improve fertility in multiple genetic backgrounds. First, we show that loss of RNA editing by ADR-2 restores normal embryo production to subfertile animals that transgenically express a vitellogenin (yolk protein) fusion to green fluorescent protein. Using this phenotype, a high-throughput screen was designed to identify RNA binding proteins that when depleted yield synthetic phenotypes with loss of adr-2. The screen uncovered a genetic interaction between ADR-2 and SQD-1, a member of the heterogenous nuclear ribonucleoprotein (hnRNP) family of RNA binding proteins. Microscopy, reproductive assays, and high-throughput sequencing reveal that sqd-1 is essential for the onset of oogenesis and oogenic gene expression in young adult animals, and that loss of adr-2 can counteract the effects of loss of sqd-1 on gene expression and rescue the switch from spermatogenesis to oogenesis. Together, these data demonstrate that ADR-2 can contribute to the suppression of fertility and suggest novel roles for both RNA editing-dependent and independent mechanisms in regulating embryogenesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/565157v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@14a3449org.highwire.dtl.DTLVardef@aead7dorg.highwire.dtl.DTLVardef@1fff947org.highwire.dtl.DTLVardef@46d487_HPS_FORMAT_FIGEXP M_FIG Graphical AbstractFigure created with BioRender. C_FIG Article SummaryThe molecular information that controls early development is RNA. Interactions between RNA and RNA binding proteins (RBPs) are critical for successful reproduction across species. In this study, we uncovered two roles for the RBP ADR-2 in regulating reproduction. First, we demonstrate that the RNA editing activity of ADR-2 regulates fertility. Next, we screened over 250 other RBPs revealed a genetic interaction between ADR-2 and SQD-1. Further analysis revealed that sqd-1 is essential for the onset of oogenesis, and an editing-independent function of ADR-2 influences proper germline gene expression and oocyte fate in these animals.

genetics↗

bioRxiv2023

ADBP-1 regulates ADR-2 nuclear localization to control editing substrate selection

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by ADAR enzymes, is a prevalent and conserved RNA modification. While A-to-I RNA editing is essential in mammals, in Caenorhabditis elegans, it is not, making them invaluable for RNA editing research. In C. elegans, ADR-2 is the sole catalytic A-to-I editing enzyme, and ADR-1 is an RNA editing regulator. ADAR localization is well-studied in humans but not well-established in C. elegans. In this study, we examine the cellular and tissue-specific localization of ADR-2. We show that while ADR-2 is present in most cells in the embryo, at later developmental stages, its expression is both tissue- and cell-type-specific. Additionally, both ADARs are mainly in the nucleus. ADR-2 is adjacent to the chromosomes during the cell cycle. We show that the nuclear localization of endogenous ADR-2 depends on ADBP-1, not ADR-1. In adbp-1 mutant worms, ADR-2 is mislocalized, while ADR-1 is not, leading to decreased editing levels and de-novo editing, mostly in exons, suggesting that ADR-2 is also functional in the cytoplasm. Besides, mutated ADBP-1 affects gene expression. Furthermore, we show that ADR-2 targets adenosines with different surrounding nucleotides in exons and introns. Our findings indicate that ADR-2 cellular localization is highly regulated and affects its function.

cell biology↗

bioRxiv2023

ADARs regulate cuticle collagen expression to promote survival to pathogen infection

BackgroundIn all organisms, the innate immune system defends against pathogens through basal expression of molecules that provide critical barriers to invasion and inducible expression of effectors that combat infection. The adenosine deaminase that act on RNA (ADAR) family of RNA binding proteins has been reported to influence innate immunity in metazoans. However, studies on the susceptibility of ADAR mutant animals to infection are largely lacking. ResultsHere, by analyzing adr-1 and adr-2 null mutants in well-established slow-killing assays, we find that both Caenorhabditis elegans ADARs are important for organismal survival to gram-negative and gram-positive bacteria, all of which are pathogenic to humans. Furthermore, our high-throughput sequencing and genetic analysis reveal that ADR-1 and ADR-2 function in the same pathway to regulate collagen expression. Consistent with this finding, our scanning electron microscopy studies indicate adr-1;adr-2 mutant animals also have altered cuticle morphology prior to pathogen exposure. ConclusionsOur data uncover a critical role of the C. elegans ADAR family of RNA binding proteins in promoting cuticular collagen expression, which represents a new post-transcriptional regulatory node that influences the extracellular matrix. In addition, we provide the first evidence that ADAR mutant animals have altered susceptibility to infection with several opportunistic human pathogens, suggesting a broader role of ADARs in altering physical barriers to infection to influence innate immunity.

molecular biology↗

bioRxiv2022

The RNA binding protein Swm is critical for Drosophila melanogaster intestinalprogenitor cell maintenance

The regulation of stem cell survival, self-renewal, and differentiation is critical for the maintenance of tissue homeostasis. Although the involvement of signaling pathways and transcriptional control mechanisms in stem cell regulation have been extensively investigated, the role of post-transcriptional control is still poorly understood. Here we show that the nuclear activity of the RNA-binding protein Second Mitotic Wave Missing (Swm) is critical for Drosophila intestinal stem cells (ISCs) and their daughter cells, enteroblasts (EBs), to maintain their identity and function. Loss of swm in these intestinal progenitor cells leads ISCs and EBs to lose defined cell identities, fail to proliferate, and detach from the basement membrane, resulting in severe progenitor cell loss. swm loss further causes nuclear accumulation of poly(A)+ RNA in progenitor cells. Swm associates with transcripts involved in epithelial cell maintenance and adhesion, and the loss of swm, while not generally affecting the levels of these Swm-bound mRNAs, leads to elevated expression of proteins encoded by some of them, including the fly orthologs of Filamin and Talin. Taken together, this study indicates a role for Swm in adult stem cell maintenance, and raises the possibility that nuclear post-transcriptional gene regulation plays vital roles in controlling adult stem cell maintenance and function.

genetics↗

bioRxiv2018

Disruption in A-to-I editing levels affects C. elegans development more than a complete lack of editing

A-to-I RNA editing is widespread in eukaryotic transcriptomes and plays an essential role in the creation of proteomic and phenotypic diversity. Loss of ADARs, the proteins responsible for A-to-I editing, results in lethality in mammals. In C. elegans, knocking out both ADARs, ADR-1 and ADR-2, results in aberrant behavior and abnormal development. Studies have shown that ADR-2 can actively deaminate dsRNA while ADR-1 cannot. However, as most studies of C. elegans ADARs were performed on worms mutated in both ADAR genes, the effects observed cannot be attributed to a single ADAR or to the interactions between ADAR genes. Therefore, we set to study the effects of each C. elegans ADAR on RNA editing, gene expression, protein levels and the contribution of each of ADAR to the phenotypes observed in worms mutated in both genes, in order to elucidate their distinct functions. We found significant differences in the phenotypes observed in worms mutated in a single ADAR gene. Worms harboring adr-1 mutations have a significant reduction in their lifespan, while worms harboring adr-2 mutations have extended lifespan. We also observed severe abnormalities in vulva formation in adr-1 mutants, and we suggest that these phenotypes are a result of an RNA editing independent function of ADR-1. Mutations in each ADAR resulted in expressional changes in hundreds of genes, and a significant downregulation of edited genes. However, very few changes in the protein levels were observed. In addition, we found that ADR-1 binds many edited genes and primarily promotes editing at the L4 stage of development. While editing still occurs in the absence of ADR-1, most of the editing occurs in genes that are edited in wildtype worms, suggesting that ADR-1 does not prevent editing by binding to and protecting the RNA but rather enhances or promotes editing. Our results suggest that ADR-1 plays a significant role in the RNA editing process and by altering editing levels it causes the severe phenotypes that we observed. In contrast, a complete lack of RNA editing is less harmful to the worms. Furthermore, our results indicate that the effect of RNA editing on the protein content in the cell is minor and probably the main purpose of these modifications is to antagonize or enhance other gene regulatory mechanisms that act on RNA.

genomics↗