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.

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?

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.

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