Biology subjects

Rozen-Gagnon, K.

Publications and source records attributed to Rozen-Gagnon, K..

5 recordsLinked to original sources

bioRxiv2025

Heterozygosity at a conserved candidate sex determination locus is associated with female development in the clonal raider ant

Sex determination is a developmental switch that triggers sex-specific developmental programs. This switch is "flipped" by the expression of genes that promote male- or female-specific development. Many lineages have evolved sex chromosomes that act as primary signals for sex determination. However, haplodiploidy (males are haploid and females are diploid), which occurs in ca. 12% of animal species, is incompatible with sex chromosomes. Haplodiploid taxa must, therefore, rely on other strategies for sex determination. One mechanism, "complementary sex determination" (CSD), uses heterozygosity as a proxy for diploidy. In CSD, heterozygosity at a sex determination locus triggers female development, while hemizygosity or homozygosity permits male development. CSD loci have been mapped in honeybees and two ant species, but we know little about their evolutionary history. Here, we investigate sex determination in the clonal raider ant, Ooceraea biroi. We identified a 46kb candidate CSD locus at which all females are heterozygous, but most diploid males are homozygous for either allele. As expected for CSD loci, the candidate locus has more alleles than most other loci, resulting in a peak of nucleotide diversity. This peak negligibly affects the amino acid sequences of protein-coding genes, suggesting that heterozygosity of a non-coding genomic sequence triggers female development. This locus is distinct from the CSD locus in honeybees but homologous to a CSD locus mapped in two distantly related ant species, implying that this molecular mechanism has been conserved since a common ancestor that lived approximately 112 million years ago.

evolutionary biology↗

bioRxiv2024

Distinct chikungunya virus polymerase palm subdomains contribute to virus replication and virion assembly

Alphaviruses encode an error-prone RNA-dependent RNA polymerase (RdRp), nsP4, required for genome synthesis, yet how the RdRp functions in the complete alphavirus life cycle is not well-defined. Previous work using chikungunya virus (CHIKV) has established the importance of the nsP4 residue cysteine 483 in maintaining viral genetic fidelity. Given the location of residue C483 in the nsP4 palm domain, we hypothesized that other residues within this domain and surrounding subdomains would also contribute to polymerase function. To test this hypothesis, we designed a panel of nsP4 variants via homology modeling based on the Coxsackievirus B3 3 polymerase. We rescued each variant in both mammalian and mosquito cells and discovered that the palm domain and ring finger subdomain contribute to polymerase host-specific replication and genetic stability. Surprisingly, in mosquito cells, these variants in the ring finger and palm domain were replication competent and produced viral structural proteins, but they were unable to produce infectious progeny, indicating a yet uncharacterized role for the polymerase in viral assembly. Finally, we have identified additional residues in the nsP4 palm domain that influence the genetic diversity of the viral progeny, potentially via an alteration in NTP binding and/or discrimination by the polymerase. Taken together, these studies highlight that distinct nsP4 subdomains regulate multiple processes of the alphavirus life cycle, placing nsP4 in a central role during the switch from RNA synthesis to packaging and assembly. Author SummaryChikungunya virus (CHIKV) is a re-emerging alphavirus transmitted to humans by mosquitoes and causing frequent explosive outbreaks. Its replication relies on a polymerase that incorporates a significant number of errors in the new genomes, making it a good candidate to develop vaccines or antiviral strategies. However, little is known on alphavirus polymerase function in alternate hosts. To begin to understand how the CHIKV polymerase nsP4 functions, we designed a panel of nsP4 variants taking advantage of the conservation of polymerase structure across positive strand RNA viruses. We discovered that the palm domain and ring finger of the polymerase were involved in host-specific RNA replication, genetic stability, and virus assembly. In addition, we demonstrated that the palm domain directly impacted the generation of viral genetic diversity. Taken together, these findings add further evidence to the crucial impact of the core palm domain of CHIKV polymerase not only on the replication of the RNA itself, but also on the genetic stability of the protein, as well as its involvement in viral assembly.

microbiology↗

bioRxiv2023

Mapping RNA-Protein Interactions with Subcellular Resolution Using Colocalization CLIP

RNA binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP, a method that combines CrossLinking and ImmunoPrecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the well-studied RNA-binding protein HuR. Using this method, we uncover HuRs dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule compartments. We uncover HuRs unique binding preferences within stress granules during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP:RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.

molecular biology↗

bioRxiv2020

A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines

Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~700,000 deaths each year. Genetic mutants are valuable tools to interrogate both fundamental vector biology and mosquito host factors important for viral infection. However, very few genetic mutants have been described in mosquitoes in comparison to model organisms. The relative ease of applying CRISPR/Cas9 based gene editing has transformed genome engineering and has rapidly increased the number of available gene mutants in mosquitoes. Yet, in vivo studies may not be practical for screening large sets of mutants or possible for laboratories that lack insectaries. Thus, it would be useful to adapt CRISPR/Cas9 systems to common mosquito cell lines. In this study, we generated and characterized a mosquito optimized, plasmid based CRISPR/Cas9 system for use in U4.4 (Ae. albopictus) and Aag2 (Ae. aegypti) cell lines. We demonstrated highly efficient editing of the AGO1 locus and isolated knock-down AGO1 cell lines. Further, we used homology-directed repair to establish knock-in Aag2 cell lines with a 3xFLAG-tag at the N-terminus of endogenous AGO1. These experimentally verified plasmids are versatile, cost-effective, and efficiently edit immune competent mosquito cell lines that are widely used in arbovirus studies.

molecular biology↗

bioRxiv2020

TMEM41B is a pan-flavivirus host factor

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection. Based on our mechanistic studies we hypothesize that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication. HIGHLIGHTSTMEM41B and VMP1 are required for both autophagy and flavivirus infection, however, autophagy is not required for flavivirus infection. TMEM41B associates with viral proteins and likely facilitates membrane remodeling to establish viral RNA replication complexes. TMEM41B single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection. TMEM41B-deficient cells display an exaggerated innate immune response upon high multiplicity flavivirus infection.

molecular biology↗