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Reposted by Sara Rouhanifard

bioRxivpreprint @biorxivpreprint.bsky.social · 7d

Multi-Scale Kinetics Modeling and Advanced Assay for mRNA-Lipid Nanoparticle Potency Assessment https://www.biorxiv.org/content/10.1101/2025.09.29.679406v1

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Reposted by Sara Rouhanifard

STAR Protocols @cp-starprotocols.bsky.social · Jul 25

Protocol for differential analysis of pseudouridine modifications using nanopore DRS and unmodified transcriptome control #protocol #starprotocols #cellpress

Protocol for differential analysis of pseudouridine modifications using nanopore DRS and unmodified transcriptome control

In this protocol, we detect pseudouridine using nanopore direct RNA sequencing (DRS 002/004) and analyze dynamic changes to mRNA modifications in response to perturbation. We cover RNA extraction, poly(A) selection, DRS, unmodified transcriptome (in vitro transcribed [IVT]) library preparation, and knockdowns of writer proteins as critical controls. We detail preprocessing, psi-site identification, and differential analysis, quantifying the direction and magnitude of changes across cellular type...

dlvr.it

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Reposted by Sara Rouhanifard

RNA Society Journal @rnajournal.bsky.social · Aug 5

RNA modifications in T cells are more stable than expected—primary and immortalized cells share most sites, suggesting common regulatory features can be studied across systems bit.ly/3GP97C8

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Sara Rouhanifard @srouhanifard.bsky.social · Jul 17

Many thanks to the team!! 🤓🤓🤡🥳 @sashafanari.bsky.social @wanunupore.bsky.social

Sara Rouhanifard @srouhanifard.bsky.social · Jul 17

5/
So are immortalized cells “damaged”? Not quite.
They’re not perfect stand-ins, but they’re surprisingly consistent with primary cells.
We conclude that Jurkat cells are a solid model for studying ψ — just watch out for that 13%.
📖 tinyurl.com/TCellPsiRNA

RNA | Mobile

tinyurl.com

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Sara Rouhanifard @srouhanifard.bsky.social · Jul 17

4/
The twist? It’s not due to differences in pseudouridine synthase expression — enzyme levels were similar.
So what's driving site selection? Likely trans-regulatory factors or RNA structure, not just enzyme abundance.

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Sara Rouhanifard @srouhanifard.bsky.social · Jul 17

3/ But that other 13%?
☑️ Jurkat-specific ψ-sites hit oncogenic and immune activation genes
☑️ Primary T cell–specific ψ-sites appear on trafficking and calcium signaling genes
🧬 Immortalized cells also showed more clustered ψ patterns, hinting at altered control compared to primary T cells.

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Sara Rouhanifard @srouhanifard.bsky.social · Jul 17

2/ We were genuinely surprised by this result — we expected transformation to cause widespread disruption. But 87% of ψ-sites were shared between Jurkat and primary T cells!
Most differences came from gene expression, not ψ-site selection.
This process seems to be tightly conserved.

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Sara Rouhanifard @srouhanifard.bsky.social · Jul 17

1/🧵
🚨 New paper published in RNA!
Scientists often say anecdotally that RNA modifications are disrupted in immortalized cells — but no one’s really tested it.
So we did: ψ-mapping in primary T cells vs. Jurkat cells using direct RNA-seq.
📄 tinyurl.com/TCellPsiRNA
#nanopore #RNA #pseudouridine

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Reposted by Sara Rouhanifard

Peng Wu @pengwu.bsky.social · Jun 17

In this incredible work, @srouhanifard.bsky.social developed a new catalyst, which makes CuAAC truly biocompatible
rdcu.be/enfHz

inCu-click: DNA-enhanced ligand enables live-cell, intracellular click chemistry reaction with copper catalyst

Nature Communications - Tracking biomolecules in living cells requires fast but gentle labeling. Here, authors introduce inCu-click, a DNA-conjugated ligand that localizes copper to enable...

rdcu.be

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Sara Rouhanifard @srouhanifard.bsky.social · May 29

Just published: “inCu-click: DNA-enhanced ligand enables live-cell click chemistry with copper catalyst” in @natcomms.nature.com! 🚀

A DNA-conjugated chelator locks Cu⁺ ions for in situ CuAAC at nanomolar doses—zero toxicity.

Read the full paper here ➡️ rdcu.be/enfHz #clickchemistry

inCu-click: DNA-enhanced ligand enables live-cell, intracellular click chemistry reaction with copper catalyst

Nature Communications - Tracking biomolecules in living cells requires fast but gentle labeling. Here, authors introduce inCu-click, a DNA-conjugated ligand that localizes copper to enable...

rdcu.be

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Sara Rouhanifard @srouhanifard.bsky.social · Mar 21

6/6 🙌 Future work will determine whether static sites are critical for cellular function while plastic sites fine-tune gene expression in response to environmental stressors.
Huge thanks to the team for all their hard work! @wanunupore.bsky.social @genometdcc.bsky.social

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Sara Rouhanifard @srouhanifard.bsky.social · Mar 21

5/6 ⚙️ Enzyme insights: TRUB1 and PUS7 levels shifted with differentiation, and KD experiments reveal unexpected coregulation—knocking down one lowers Ψ at the other’s targets. This hints at a coordinated network fine-tuning mRNA Ψ for neuronal homeostasis and stress resilience.

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Sara Rouhanifard @srouhanifard.bsky.social · Mar 21

4/6 📊 Plasticity vs stability: ~30% of Ψ sites changed occupancy across conditions (“plastic”), while the rest remained constant (“static”). Among the plastic sites was a key Ψ site in YTHDF1—an m6A reader involved in translation, which changed in response to cellular and environmental cues.

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Sara Rouhanifard @srouhanifard.bsky.social · Mar 21

3/6 📊 Key finding #1: Pb²⁺ treated cells had more Ψ sites transcriptome-wide but lower relative occupancy per site vs untreated/differentiated cells—suggesting a broad but shallow protective pseudouridylation response to stress.

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Sara Rouhanifard @srouhanifard.bsky.social · Mar 21

2/6 🧠Link here: authors.elsevier.com/a/1koBC8YyDf...
Method: SH SY5Y cells were untreated, differentiated with retinoic acid, or exposed to Pb²⁺. Nanopore DRS determined site-specific, relative Ψ “occupancy”; orthogonal knockdowns (TRUB1, PUS7) and biochemical assays validated sites.

authors.elsevier.com

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Sara Rouhanifard @srouhanifard.bsky.social · Mar 21

1/6 🧬New paper at @cp-cellsystems.bsky.social led by @sashafanari.bsky.social! Does pseudouridine (Ψ) dynamically respond to cell state? We used nanopore DRS + Mod-p ID to map Ψ in neuron-like cells under “normal” and “perturbed” conditions (healthy differentiation and unhealthy Pb2+ poisoning).

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Reposted by Sara Rouhanifard

Arjun Raj @arjunraj.bsky.social · Mar 18

Sitting in a journal club-style class right now and after the main presenters present, there are "roles" from others in the group, like the "archeologist" who looks into the citation trail, the "private investigator" who tries to dig into the history, and the "industry practitioner", etc. Cool idea!

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Reposted by Sara Rouhanifard

GenomeTDCC @genometdcc.org · Mar 10

🧪🧬👩‍🔬🖥️
Do immortalized cells have a different RNA modification profile compared to primary cells? 🤔

@sashafanari.bsky.social @wanunupore.bsky.social @srouhanifard.bsky.social assess the most abundant RNA modification, pseudouridine, in primary and immortalized human T cells
👇
doi.org/10.1101/2025...

Direct RNA sequencing of primary human T cells reveals the impact of immortalization on mRNA pseudouridine modifications.

Immortalized cell lines are commonly used as proxies for primary cells in human biology research. For example, Jurkat leukemic T cells fundamentally contributed to uncovering T cell signaling, activat...

doi.org

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Reposted by Sara Rouhanifard

bioRxiv Genomics @biorxiv-genomic.bsky.social · Mar 10

Direct RNA sequencing of primary human T cells reveals the impact of immortalization on mRNA pseudouridine modifications. https://www.biorxiv.org/content/10.1101/2025.03.02.641090v1