Dan Depledge

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!

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 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…

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

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.

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?

Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing - Nature Biotechnology tRNA abundance and chemical modifications are measured simultaneously with nanopores.

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…

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.

Defining expansions and perturbations to the RNA polymerase III transcriptome and epitranscriptome by modified direct RNA nanopore sequencing RNA polymerase III (Pol III) transcribes cytosolic transfer RNAs (tRNAs) and other non-coding RNAs (ncRNAs) essential to cellular function. However, many aspects of Pol III transcription and processin...

Our latest preprint is online!

In which we take a left turn and dive head first into the biology of the RNA polymerase III transcriptome and epitranscriptome

Do you want to know more? A small thread follows👇

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

Post‐Transcriptional Regulation of Gene Expression and the Intricate Life of Eukaryotic mRNAs The central dogma of biology relies on numerous regulatory events to define the expression profile of any given cell. Created in https://BioRender.com.

#GeneExpression #Review #mRNA #RNA #RNAProcessing

wires.onlinelibrary.wiley.com/doi/full/10....

A teaser for our new preprint that will be available soon!!!

Pol III, RNA modifications, tRNAs, viruses, and of course, nanopores

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...

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

Repression of varicella zoster virus gene expression during quiescent infection in the absence of detectable histone deposition Author summary Varicella zoster virus (VZV) establishes latent infection exclusively in human neurons. This phase of infection is characterized by restricted viral gene expression, lack of detectable ...

Happy to share our publication in #PlosPathogens investigating repression of varicella zoster virus in neuron-like cells: journals.plos.org/plospathogen...
Thanks to @dandepledge.bsky.social and other colleagues for their contribution!

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!

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

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)_/¯

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: ???

today has been cancelled go back to bed ALT: today has been cancelled go back to bed

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!!!

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

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...

Making progress...

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

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

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

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

It always rains in Hannover...

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