Sara Rouhanifard

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

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...

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

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

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

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

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.

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.

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.

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

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...

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...

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

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

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.

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.

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.

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.

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).

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!

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...

🧪🧬👩‍🔬🖥️
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. https://www.biorxiv.org/content/10.1101/2025.03.02.641090v1

Office hours are starting

Identification of molecular determinants of gene-specific bursting patterns by high-throughput imaging screens Sood et al. used high-throughput screening to identify regulators of gene bursting and find both gene- and cell-type-specific effectors. Acetylation alters burst size in a gene-specific manner but is independent of promoter acetylation. Rather,…

Online Now: Identification of molecular determinants of gene-specific bursting patterns by high-throughput imaging screens

Single-molecule live-cell RNA imaging with CRISPR–Csm - Nature Biotechnology Single-molecule live-cell fluorescence in situ hybridization uses an RNA-targeting CRISPR–Csm complex to image and track endogenous RNAs.

Single-molecule live-cell RNA imaging with CRISPR–Csm - @doudna-lab.bsky.social @qb3-berkeley.bsky.social @innovativegenomics.bsky.social go.nature.com/3EJPKsM

I sat on a study section that met this past Friday (2/14)

Protein codes promote selective subcellular compartmentalization Cells have evolved mechanisms to distribute ~10 billion protein molecules to subcellular compartments where diverse proteins involved in shared functions must assemble. Here, we demonstrate that prote...

Cool paper using LLM to discover a protein sequence code for subcellular localization 👏

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

Gene editing technology began by people studying salt marshes. Ozempic began by folks studying the venom of Gila Monsters. Support for basic science has empowered us to understand our world. Tethering it to applications health has transformed and saved countless lives.

Graphic showing modification of mRNA with ADP-ribose mediated by bacterial enzyme cmdTAC

Check it out! Another novel RNA modification - ADP-ribosylation - that was previously only known on proteins. A cousin to #glycoRNA 👏

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

"Imagine a DAPI-like stain, but for the extracellular matrix." That's basically how this work was pitched to me by Kayvon and Antonio a year or so ago. Now the final product really delivers. Read about their versatile label for ECM in living tissues here: www.nature.com/articles/s41...

NuclampFISH enables cell sorting based on nuclear RNA expression for chromatin analysis Transcriptional bursts refer to periods when RNA polymerase interacts with a DNA locus, leading to active gene transcription. This bursting activity can vary across individual cells, and analyzing the...

Liu, ... Rouhanifard have optimized nuclear FISH to the point where it's now possible to sort cells based on transcript production. They sort cells into bins and measure chromatin state at the locus whose tx was sorted on.
www.biorxiv.org/content/10.1...