iiinova — where your digital self keeps evolving

Finally, we show that infection with HSV-1 perturbs the Pol III transcriptome and epitranscriptome, increasing the relative abundances of pre-tRNAs while reducing rates of pseU installation. I for one am now excited to see where this all goes next!!! Thanks for reading!

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We show that pseudouridine (pseU) modifications are common markers of Pol III transcribed RNAs and provide a detailed analysis of pseU installation across pre-tRNAs. When combining DRAP3R and nano-tRNA-Seq, we are able to distinguish RNA mods installed co- versus post-transcriptionally.

Evidence of RNA polymerase III recruitment and transcription at protein-coding gene promoters

And so, what did we learn from DRAP3R? Firstly, we show this is a robust, sensitive and reproducible method. We also confirmed the existence of several recently described putative Pol III transcribed RNAs (www.sciencedirect.com/science/arti...) and identified several more candidates

sciencedirect.com

The transcriptional interplay of human RNA polymerase I (RNA Pol I), RNA Pol II, and RNA Pol III remains largely uncharacterized due to limited integr…

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This decision has been validated many times over as the new chemistry allows the sequencing of RNAs as short as 20nt and enables native detection of RNA modifications such as m6A and pseudouridine which enabled us to further broaden our analyses.

Given the time it takes to publish, we reasoned that our method would be rendered obsolete if we stuck to the RNA002 chemistry and so we made the difficult/frustrating/expensive decision to drop all our existing datasets and instead switch to the new chemistry and generate new data.

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In Oct 2023, ONT announced that the RNA002 chem would be discontinued by end of year and replaced with a new (RNA004) chem would improve yield and accuracy. This led to a significant dilemma – do we continue working with the existing chem or drop everything we had so far and switch to the new one?

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Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing - Nature Biotechnology

Morghan Lucas and the Novoa lab published a game-changing paper on how to directly sequence mature tRNAs using nanopore DRS (www.nature.com/articles/s41...). Implementing the suggested changes worked brilliantly and we could now capture large numbers of diverse Pol III transcribed RNAs. But then…

nature.com

tRNA abundance and chemical modifications are measured simultaneously with nanopores.

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However, while our early results were promising, this was still in the era of the RNA002 chemistry from nanopore which had a number of limitations, particularly in regards to sequencing shorter RNAs. This led to sequencing runs missing large numbers of RNAs (inc. tRNAs)… But then came a solution!

And so, DRAP3R was born! DRAP3R takes advantage of the fact that all nascent Pol III transcribed RNAs have short (4-6 nt) template-derived poly(U) tracts at the 3’ end and these RNAs are theoretically possible to capture using a modified nanopore DRS reverse transcription adaptor.

Given our interest relates to how Pol III biology is influenced by viral infections, not having a good method to read out changes is rather frustrating. And so, after lots of discussion with my senior postdoc (Ruth Verstraten), we decided that, as always, nanopore sequencing is the answer!

Particular highlights were these three papers genome.cshlp.org/content/20/6... www.nature.com/articles/s41... www.jci.org/articles/vie... It was clear from these (and other) papers that it is very hard to define and measure changes to the Pol III transcriptome.

A benefit to moving from NYU to the Hannover Medical School to try and set up a new lab in the middle of a pandemic was that I had a lot(!) of time to read. As in months and months. This enabled me to reengage in my long-standing fascination in Pol III biology.

Dan Depledge @dandepledge.bsky.social · 07/03/2025

A teaser for our new preprint that will be available soon!!! Pol III, RNA modifications, tRNAs, viruses, and of course, nanopores

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Reposted by Dan Depledge

Exzellenzcluster RESIST @resist-cluster.bsky.social · 12/02/2025

The master’s program in #biomedical_data_science, which was developed as part of RESIST, has now graduated for the second time: 18 students from the first two years successfully completed their studies. www.resist-cluster.de/en/festive-s... 🎉 @dandepledge.bsky.social @bosse-lab.bsky.social

resist-cluster.de

Festive setting

Festive setting for the presentation of certificates to students of biomedical data science: The master's program in biomedical data science, which was developed as part of the RESIST project, has now...

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Dan Depledge @dandepledge.bsky.social · 06/02/2025

Congratulations to all the graduates of the MHH Biomedical Data Science Masters programme! The graduation ceremony today was a great success and we really appreciated @bosse-lab.bsky.social giving a great lecture!

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Dan Depledge @dandepledge.bsky.social · 29/01/2025

Love it when you read a review that completely unlocks a new field of biology that you are working in.

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Dan Depledge @dandepledge.bsky.social · 08/01/2025

admin: send us all your registration documents and passports because we forgot to put these on file when you joined. admin: this is also your fault... me: ??? admin: when you have resolved all these issues, please inform us of how you did it. me: ¯\_(ツ)_/¯

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Dan Depledge @dandepledge.bsky.social · 08/01/2025

me: i need help with some visa questions local admin directly responsible for such matters: no me: ??? admin: you need to sort this out yourself admin: also, you are not properly registered as a foreigner in our system, this is clearly your fault and definitely not ours... me: ???

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Dan Depledge @dandepledge.bsky.social · 07/01/2025

First day back at work and so far I have a broken computer, a VPN that cannot maintain it's connection and half my lab has been lost to a time management course that, no kidding, is 8 hours long!!!

media.tenor.com

today has been cancelled go back to bed

ALT: today has been cancelled go back to bed

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Dan Depledge @dandepledge.bsky.social · 03/01/2025

Thank goodness for section 81 paragraph 4 of the German residence act!

Dan Depledge @dandepledge.bsky.social · 02/01/2025

2025 goals Get off facebook - done Get off twitter/x - done Navigate German bureaucracy to obtain a settlement permit so that I am not forced to leave the EU in June - pending Brexit, truly the gift that keeps on giving...

Dan Depledge @dandepledge.bsky.social · 17/11/2024

So good to see membership of this platform increasing so fast. So much great science content already :)

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Dan Depledge @dandepledge.bsky.social · 11/11/2024

Nice to see the communities of RNA biologists and herpesvirologists are growing on here. Now we just need more nanopore people!

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Dan Depledge @dandepledge.bsky.social · 13/11/2023

Might be affordable but doesn't count for much if they keep closing classes and sending kids home at very short notice...

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Dan Depledge @dandepledge.bsky.social · 13/11/2023

The tweet or the weather? Hoping I don't have to make an obscure joke about Anders Celsius ;)

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Dan Depledge @dandepledge.bsky.social · 13/11/2023

Remind me again why I left the wonderful sun and blue skies of NYC...

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Marco Galardini @mgalactus.bsky.social · 06/11/2023

The HZI will hire up to twenty (20!) tenure-track research groups under the broad topic of "Microbial Stargazing" (🦠🌌). The first round of 4 positions is now open; the call closes on the 10th of December. Please share! www.helmholtz-hzi.de/en/career/jo...

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Dan Depledge @dandepledge.bsky.social · 02/11/2023

Trying to write computational/sequencing focused paper that explores the biology of three very different viruses is proving a bigger challenge than I had anticipated...

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Dan Depledge @dandepledge.bsky.social · 17/10/2023

Great to see this online and very happy to have contributed to this study led by Hannah Burgess (NYU & University of Surrey) and Ian Mohr (NYU)!!! www.embopress.org/doi/full/10.... #is_there_anything_nanopores_cant_do

embopress.org

EMBO Press is an editorially independent publishing platform for the development of EMBO scientific publications.

Dan Depledge @dandepledge.bsky.social · 14/10/2023

It's been known for a good while (pubmed.ncbi.nlm.nih.gov/32313079/) which is why when looking at CoVs we supplemented meRIPSeq with an orthologous approach (comparative nanopore sequencing using a small molecule METTL3 inhibitor). Still found a number of coinciding results...

pubmed.ncbi.nlm.nih.gov

Limits in the detection of m6A changes using MeRIP/m6A-seq - PubMed

Many cellular mRNAs contain the modified base m6A, and recent studies have suggested that various stimuli can lead to changes in m6A. The most common method to map m6A...

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Dan Depledge @dandepledge.bsky.social · 14/10/2023

Very much looking forward to reading this, especially with coronaviruses in mind as a number of studies (inc one I contributed to) have reported varying levels of m6A to be present (validated by orthogonal methods) which remains counterintuitive in many ways...

Ed Emmott @edemmott.bsky.social · 27/09/2023

Reposted by Dan Depledge

The lab has just been awarded an 8-year grant to study coronavirus replication! 🍾🎉 Job ads will appear around end October but we are particularly looking for PDRAs with coronavirus reverse genetics experience or CL3 coronavirus-host interactions. ~Mar 2024 start.

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Dan Depledge @dandepledge.bsky.social · 01/10/2023

With over 100 delegates from 33 institutions across six countries, we managed to squeeze in 37 presentations and a fun social evening with many fruitful discussions and a few new collaborative ventures!

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Dan Depledge @dandepledge.bsky.social · 01/10/2023

Still recovering from the mini herpesvirus conference (+ assorted bugs the kids brought home from daycare) but still happily reflecting on what an interesting and fun day it was!!! Lots of thanks to go around but I would particularly highlight our two plenary speakers and our army of volunteers!

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Dan Depledge @dandepledge.bsky.social · 29/09/2023

The 16th mini herpesvirus workshop is in full swing! Over 100 participants from 33 institutions across six countries. An excellent plenary talk from Hannah Burgess (University of Surrey) on the role of the CCR4-NOT complex in HCMV infections (that has just been accepted for publication)!

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Dan Depledge @dandepledge.bsky.social · 27/09/2023

Easily one of my favorite books ever!

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Dan Depledge @dandepledge.bsky.social · 27/09/2023

Two days until the 16th mini herpesvirus workshop!

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Reposted by Dan Depledge

Nandan Gokhale @nandangokhale.bsky.social · 26/09/2023

m7GpppNm - VERY EXCITED to share my postdoctoral work in @ramlabuw.bsky.social’s lab, now @bioRxiv. We find that cellular RNA interacts with MAVS, the adaptor protein of the RIG-I-like receptor pathway, to promote antiviral signaling. A 🧵:

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Luis M @luismschang.bsky.social · 26/09/2023

Hold your excitement.... for now 🙃

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Dan Depledge @dandepledge.bsky.social · 23/09/2023

This is insane... and explains a lot! community.nanoporetech.com/posts/flow-c...