Fletcher Lab

Post 5/5 🧵
IMPACT: This transforms tick-borne disease research:
🎯 New drug targets
🎯 Understanding vector competence
🎯 Virus-tick evolution
Testing if mechanisms work across other tick viruses next.

👨‍🔬 collaborative project with @alf-castello.bsky.social and @alainkohlvirology.bsky.social

Identification of RNF114 as ADPr-Ub reader through non-hydrolysable ubiquitinated ADP-ribose - Nature Communications Deltex E3s modify ADP-ribosylated targets with ubiquitin, creating a hybrid modification whose readers remains unknown. Here, the authors synthesise a non-hydrolysable probe that mimics the modificati...

A few years back we discovered a dual hybrid protein modification composed of ADP-ribose dinucleotide and ubiquitin (ADPr-Ub). Now we reveal that ADPr-Ub can be further ubiquitinated by the E3 ubiquitin ligase RNF114!
www.nature.com/articles/s41...

www.science.org/doi/10.1126/...

Nine rounds of directed evolution resulted in LcαE7 variants with increased rates of OP-hydrolysis. Three LcαE7 genes (wild-type, G137D and R9) were introduced into the genome of Drosophila. Fluorometric substrate DEUP, and insecticides Diazinon and Paraoxon are shown with the scissile bond indicated in red.

How predictable are the evolutionary pathways leading to insecticide resistance? Why do some species gain resistance while others do not? We explore these questions in our latest preprint. A huge effort from Rebecca Frkic, Alex Giang, Colin Jackson and the team.

www.biorxiv.org/content/10.1...

PhD student in Biochemistry - Uppsala University PhD student in Biochemistry, Disciplinary Domain of Science and Technology, Faculty of Chemistry, Department of Chemistry - BMC, Uppsala University

We are looking for a PhD student in (mostly )wet-lab biochemistry to work on short linear motifs, intrinsically disordered regions, in the context of the Marie Curie network "IDPro". More info an apply here: www.uu.se/en/about-uu/...

postdoc opening in vienna: study lipid droplets (LDs) with integrative structural biology, uncover how LD proteins shuttle between cytosol & nucleus to regulate lipid metabolism, organelle architecture and immune response. #VIP3 #lipidtime #cryoEM #coffee training.vbc.ac.at/post-docs/vi...

🚨🚨 News from the @castello-lab.bsky.social and @shabazlab.bsky.social. Our work, led by Louisa Iselin, reveals pervasive changes in the RNA-bound proteome induced by interferon. #RNA, #immunity, #interferon, #host-virus www.biorxiv.org/content/10.1...

Multi-omics analysis of SFTS virus infection in Rhipicephalus microplus cells reveals antiviral tick factors - Nature Communications Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) is a deadly tick-borne virus and a growing global health threat. In this study, Petit et al. used a multi-omics approach on SFTSV-infected tic...

OMG! I can finally share my joy to see my SFTSV work published in @natcomms.nature.com! This adventure started by the award of my MSCA grant and my move to the Kohl and @brennanlab.bsky.social at @cvrinfo.bsky.social. Let's chat about how we discovered novel tick anti-viral effectors!

Happy that my work on RNF213 has been published as a part of doi.org/10.1038/s414...!

Many thanks to all the groups involved:
@clausenlab.bsky.social
@e3chembio.bsky.social
@fletcherlab.bsky.social

Have a look at the summary from Fletcher lab for an insider perspective of the paper's history:

A family of E3 ligases extend K11 polyubiquitin on sites of MARUbylation Ubiquitin (Ub) cooperation with other post-translational modifications provides a tiered opportunity for protein regulation. Small modifications to Ub such as phosphorylation, acetylation, or ADP-ribo...

Sharing the next installment of our fun collaboration with @michaelnadbio.bsky.social at the interface of #ubiquitin and #ADP-ribosylation! Building upon our identification of cellular MARUbylation, we now identify reader/writer E3 ligases that extend K11 polyUb! 🧵
www.biorxiv.org/content/10.1...

Excited to share our new paper on Nature - how Ku accommodates Alu expansion in primates by binding to dsRNA, providing a clue for both the high levels of Ku and its essentiality in human cells. Thank @chaolinzhang @hchung03 @LenaSteckelberg More to come www.nature.com/articles/s41...

Thanks to all the many who contributed along the way and the very supportive editorial team @natcomms.nature.com

(end).

Finally, we are excited by the ability to manually adjust RNF213 activity using a nucleotide. Perhaps other E3s similarly respond to – and can be controlled by – metabolic intermediates.

We speculate that the broad sensitivity of RNF213 toward different classes of intracellular pathogen (see the beautiful work from the Randow, Coers, Impens, Pan, Sibley, Thurston and Pruneda labs) could be facilitated by cellular energy changes following IFN signaling.

Juraj and @dangrabarczyk.bsky.social solved yet more cyro-EM structures, among which they defined the cryptic E2 binding site (spoiler: no RINGs). I find it amusing (and telling) that RNF213 was only annotated as an E3 by virtue of its RING, which so far lacks well-defined function.

Meanwhile, @juraj-ahel.bsky.social had established that ATP binding, rather than hydrolysis, was key. Using the ABP, Juraj had already mapped the catalytic cysteine to a short zinc-coordinating loop, the RNF213-ZNFX1 (RZ) fold. So, the AAA and E3 modules were mechanistically connected.

In parallel experiments, we’d stumbled upon an observation, made by others too, that IFNs increase cellular ATP levels. ABP electroporation revealed that endogenous RNF213 E3 activity was coupled to such energy changes.

Once up and running, new postdoc @ardabalci.bsky.social and I began playing with introducing activity-based probes into cells, to monitor E3 activity without first lysing the culture. This led to the first surprise that one could manually alter RNF213 activity ‘in cellula’ by adjusting ATP levels.

Enter pandemic. Then, retrospectively, a poorly timed relocation to the CVR during the height of its COVID-response; slow work building a lab, plentiful distractions…

Around this time we were profiling E3s regulated by interferon (IFN), identifying RNF213 using a cysteine-reactive activity-based probe (ABP). We’d tried to capture an RNF213-ABP complex with ATP to little effect. @petermabbitt.bsky.social suggested non-hydrolysable analogues. Floodgates opened.

Moyamoya disease factor RNF213 is a giant E3 ligase with a dynein-like core and a distinct ubiquitin-transfer mechanism RNF213 is a giant E3 ligase with a dynein-like core and a unique ubiquitination mechanism that proceeds in a RING-independent manner and is linked with the Moyamoya disease.

Both observations formed key predictions that turned out to be true. Why a AAA ATPase had a built-in E3 was also a mystery.

elifesciences.org/articles/56185

A wee summary of our paper:

Tim and Juraj’s landmark paper from 2020 was the first to elucidate the structural and biochemical detail of RNF213, the largest known E3 and only AAA-E3 hybrid. Among many observations, two stood out: E3 activity was RING-independent and specific for the E2 UBE2L3.

ATP functions as a pathogen-associated molecular pattern to activate the E3 ubiquitin ligase RNF213 - Nature Communications RNF213 is an E3 ligase with ATPase activity. Here, the authors show that RNF213 is activated by ATP binding and senses cellular energy states, and reveal a transthiolation mechanism induced by immune ...

Great to see this out. A story that spanned continents, cities and budget codes…

www.nature.com/articles/s41...

Big thanks to all the teams involved!

@clausenlab.bsky.social @e3chembio.bsky.social

Omics analyses uncover host networks defining virus-permissive and -hostile cellular states The capacity of host cells to sustain or restrict virus infection is influenced by their proteome. Understanding the compendium of proteins defining cellular permissiveness is key to many questions in...

Very happy to see Honglin Chen's DPhil work and and @alf-castello.bsky.social passion project (aren't all projects passion projects in academia?) finally get published!

www.mcponline.org/article/S153...

Ubiquitin & Friends Fiesta in Vienna 2025 -- Final Call

despite these challenging times for science, we look forward to gather for a lively meeting and share the joy of discovery. to join an exciting program and connect with fellow ubiquitinists sign up at www.protein-degradation.org/symposium/

Uncovering the viral aetiology of undiagnosed acute febrile illness in Uganda using metagenomic sequencing - Nature Communications Acute febrile illness is common in sub-Saharan Africa and causative agents are often unknown. Here, the authors perform metagenomic sequencing on samples from patients with acute febrile illness in Ug...

New in Nature Comms: We applied metagenomic + targeted NGS to serum from febrile patients in Uganda. Post-COVID, LMICs have sequencing infrastructure that could be repurposed for pathogen discovery.
🔗 www.nature.com/articles/s41...

Comparative study of tropism and emergence of virulent companion animal viruses using AI-powered text mining at University of Glasgow on FindAPhD.com PhD Project - Comparative study of tropism and emergence of virulent companion animal viruses using AI-powered text mining at University of Glasgow, listed on FindAPhD.com

Come do a PhD with me using LLMs and comparative methods to understand the emergence of companion animal viruses! 🧠☠️🕸️🐕🐈🖥️

Fully funded 3.5 year PhD based at @sbohvm.gla.ac.uk and @cvrinfo.bsky.social supervised by myself, Margaret Hosie, and Willie Weir

www.findaphd.com/phds/project...

Online now! @pottslab.bsky.social @amgen.bsky.social demonstrate proof-of-concept of disease-specific targeted degradation by redirecting virally encoded E3 ubiquitin ligases with VIPER-TACs. http://dlvr.it/TJLmvh

Alternative splicing broadens antiviral diversity at the human OAS2 locus Interferons (IFN) are cytokines that regulate the expression of hundreds of genes during viral infections to generate a broadly antiviral environment in the stimulated cell. Antiviral breadth is provi...

It’s a great pleasure to advertise the pre-print of Emma’s OAS2 story!

doi.org/10.1101/2025...

One gene, two transcripts, two antiviral molecules with different virus targets, using different antiviral mechanisms… a great place to release into the community! Well done Emma!