Biology subjects

Wagner, M. E.

Publications and source records attributed to Wagner, M. E..

3 recordsLinked to original sources

bioRxiv2025

An apicoplast localized GTPase is essential for Toxoplasma gondii survival

The apicoplast is an essential organelle found in Apicomplexa, a large phylum of intracellular eukaryotic pathogens. The apicoplast produces metabolites that are utilized for membrane biogenesis and energy production. A majority of apicoplast resident proteins are encoded by the nuclear genome and are trafficked to the apicoplast, and are referred to as Nuclear Encoded and Apicoplast Targeted (NEAT) proteins. In this study, we characterized a NEAT protein named TgBipA, that is a homolog of the highly conserved prokaryotic translational GTPase BipA. BipA is essential for bacterial survival in stress conditions and functions through interactions with the prokaryotic ribosome, although its role is not fully understood. Through genetic knockouts of TgBipA and immunofluorescence imaging we show that loss of TgBipA results in apicoplast genome replication defects, disruption of NEAT trafficking, loss of the apicoplast, and subsequently parasite death. Furthermore, we show through comparative studies that this phenotype closely resembles the delayed death phenomenon observed when inhibiting apicoplast translation. Finally, we show that TgBipA is an active GTPase in vitro, and its GTP hydrolysis activity is critical for its cellular function. Our findings demonstrate TgBipA is a GTPase which has an essential role in apicoplast maintenance, providing new insight into the cellular processes of the organelle. Importance StatementToxoplasma gondii, and many other parasites in the phylum Apicomplexa, are pathogens with significant medical and veterinary importance. Most Apicomplexa contain a non-photosynthetic plastid organelle named the apicoplast. This organelle produces essential metabolites and perturbation of apicoplast function results in parasite death. The apicoplast contains bacterial-like pathways for apicoplast genome replication and expression. Thus, the discovery of the apicoplast lead to optimism that this organelle would provide a wealth of anti-parasitic drug targets. Therefore, the identification and characterization of new apicoplast proteins could provide new opportunities for therapeutic development. In this study we characterized the function of a protein called TgBipA, a homolog of a highly conserved bacterial GTPase bipA which has been implicated in maturation of the 50S ribosomal subunit and adaptation to cellular stress. We show that TgBipA is essential for apicoplast maintenance and parasite survival.

microbiology↗

bioRxiv2025

U1 snRNP and RNA polymerase II interaction is predominantly mediated by Prp40 rather than U1-70K in yeast

Transcription and splicing are coupled both temporally and physically. A previous cryo-EM structure of the human U1 snRNP and RNA polymerase pol II complex has shown that U1 snRNP uses predominantly the RRM domain of U1-70K to directly interact with the RPB2 subunit of pol II. However, residues on U1-70K involved in the interaction with pol II are not conserved in yeast U1-70K, raising the question whether yeast U1 snRNP interacts with pol II in a similar manner. We found that yeast pol II associates with both U1 and U2 snRNPs, but U1-70K makes a minimal contribution to U1 snRNPs interaction with pol II. On the other hand, multiple domains of yeast Prp40 interact with pol II and the removal of the C-terminal domain (CTD) of pol II does not affect this interaction. Although yeast Prp40 is stably associated with U1 snRNP, its human homologs, PRPF40a and PRPF40b, are alternative splicing factors that are not integral components of U1 snRNP. This shift of function of Prp40 homologs may have led to the evolution of U1-70K to be the main interactor with pol II in the human system.

biochemistry↗

bioRxiv2021

RASopathy Mutations Demonstrate the Critical Function of the Cysteine-rich Domain in Maintaining BRAF in an Autoinhibited State

BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD participates in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well-characterized. Here, we analyze a panel of CRD mutations and show that they increase BRAF activity by relieving autoinhibition and/or enhancing PS binding, with relief of autoinhibition being the major factor determining mutation severity in zebrafish models. Further, we show that CRD-mediated autoinhibition is essential for preventing the constitutive plasma membrane localization of BRAF and increased RAS-dependent and RAS-independent function. Comparison of the BRAF- and CRAF-CRDs also indicates that the BRAF-CRD is a stronger mediator of autoinhibition and PS binding, and given the increased catalytic activity of BRAF, our studies reveal a more critical role for CRD-mediated autoinhibition in BRAF regulation.

cell biology↗