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Reposted by Matthew Taliaferro

Molecular Biology Ph.D. Program // CU Anschutz @molbcu.bsky.social · 23/09/2026

Congratulations to Molecular Biology student Katie Vaeth of Matt Taliaferro's lab @jmtali.bsky.social for a successful PhD defense!

Flyer advertising the thesis defense of Katie Vaeth, 9/22/2026

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Matthew Taliaferro @jmtali.bsky.social · 14/08/2026

This is something we are exploring!

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Matthew Taliaferro @jmtali.bsky.social · 13/08/2026

Yes we have puro-PLA evidence that the RNA is being translated in the intracellular bridge.

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

Thanks Aaron! It was great to see you at the meeting!

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

21/ 🎯 Bottom line: Net1 mRNA has to reach the midbody for cells to complete abscission efficiently, and Net1 protein is a new player in building the branched actin network that makes abscission, and faithful cell division, possible.

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

20/ We tested this by measuring Arp2/3 buildup at the midbody across our cell lines. Arp2/3 accumulation was lost in Net1 knockouts, restored by the full-length UTR rescue, and NOT restored by the ΔLE rescue, exactly what our model predicted.

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

19/ So our hypothesis is that localized Net1 mRNA leads to localized Net1 protein which activates Rac1 locally → Arp2/3 turns on → actin builds up → abscission happens. (It's amazing how often biology ends up resembling a Rube Goldberg machine).

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

18/ Abscission requires actin to build up at the cut site, and that build-up depends on a protein complex called Arp2/3. One GTPase, Rac1, is known to switch on Arp2/3.

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

17/ This version failed to rescue the abscission defect, so Net1's ability to bind GTPases is essential. Next question: how would local GTPase activity actually trigger abscission?

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

16/ So maybe a local pileup of Net1 mRNA (and therefore protein) creates a local pool of active GTPase. To test this, we rescued our knockout with a Net1 transgene that still had the LE in its UTR but carried a point mutation that stopped the protein from binding GTPases.

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

15/ Now I should tell you what Net1 protein does. It's a GEF (guanine nucleotide exchange factor) that switches on a family of proteins called Rho/Rac GTPases. These GTPases are known to control key steps of cell division and abscission.

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

14/ But what's the mechanism? Does having Net1 mRNA at the midbody actually build up more Net1 protein there? Using our knockout/rescue system: yes! Only Net1 protein made from RNA containing the LE showed up in meaningful amounts near the midbody.

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

13/ 🔑 So, to recap: an RNA gets trafficked to the midbody during cell division, and when it's missing, the midbody can't finish its job (abscission). This may be a clear-cut example where localizing a specific RNA has a defined, essential function!

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

12/ So where exactly does the cell cycle stall when Net1 RNA can't get to the midbody? Net1 knockout cells linger too long in telophase, the last stage of mitosis, pointing to a problem with abscission itself. The full-length UTR transgene fixed this. The ΔLE version didn't.

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

11/ To confirm this wasn't a fluke, we made a Net1 knockout line and rescued it with Net1 transgenes carrying either the full 3' UTR (traffics to the midbody) or a version missing the LE, "ΔLE" (doesn't traffic). Again, the LE was required for cells to divide efficiently.

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

10/ Now we finally knew what to target! We used antisense oligos (ASOs), short synthetic sequences that bind to and block a specific stretch of RNA, against the LE. They blocked Net1 RNA from reaching the midbody. And, unexpectedly, cell division slowed way down! ⏳

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

9/ One region in the middle of the UTR stood out. Oligos from it were enough on their own to send the reporter to the midbody. We called this the "localization element" (LE). Removing just this region also blocked transport, so it's both sufficient and necessary.

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

8/ Okay, but which sequences *within* that 3' UTR are doing the work? To find out, we used a massively parallel reporter assay (MPRA). We tiled ~500 short DNA pieces across the Net1 3' UTR, put each into a reporter RNA, and measured how well each one reached the midbody.

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

7/ Many "zip code" sequences that direct RNAs to specific places live in the 3' UTR, the tail end of an RNA that doesn't code for protein. We fused Net1's 3' UTR to a reporter RNA and tracked it by RT-qPCR. Net1's 3' UTR alone was enough to send the reporter to the midbody.

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

6/ Luckily, we can isolate the RNA contents of midbodies and compare them to whole cells. We and others had already found that specific RNAs are shuttled to the midbody. We started with one of the most enriched RNAs, Net1, and asked: how does it get there?

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

5/ This is a hard question to answer. There are only a handful of well-documented cases where sending one specific RNA to one specific place in the cell is actually important for that cell's function. Part of the problem: how do you even know what to disrupt?

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

4/ Some background: the midbody recruits a series of proteins to carry out abscission. But are RNA molecules recruited too? And if so, does it actually matter? Does having RNA there help the midbody do its job?

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

3/ Our lab studies how RNA molecules get trafficked to specific spots in the cell and why that matters for cell function. The Prekeris lab are experts on the midbody and how it drives cell division and abscission, the final "cut" that separates two new cells.

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Matthew Taliaferro @jmtali.bsky.social · 12/08/2026

Excited to share our newest work! We asked whether RNA molecules need to be in a specific place in the cell to do their job. We found that a single RNA must reach the midbody, the structure that pinches two dividing cells apart, for cell division to finish efficiently. 🧵⬇️

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Matthew Taliaferro @jmtali.bsky.social · 12/06/2026

There is one 5mer that is found in multiple critical elements, but we haven’t tested its importance yet. At the end of the day, I think we are going to need a structure to really know what is going on.

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Matthew Taliaferro @jmtali.bsky.social · 12/06/2026

Support windows tend to be more A/G rich than the transcriptome at large but not moreso than the rest of the element. We have some ideas as to what this may be. We didn’t try to swap support elements across genes. That’s an interesting idea.

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Matthew Taliaferro @jmtali.bsky.social · 12/06/2026

Thanks Xuebing! Hope you are well.

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Matthew Taliaferro @jmtali.bsky.social · 11/06/2026

Thanks Junjie! Hope you are well.

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Matthew Taliaferro @jmtali.bsky.social · 11/06/2026

We also wonder that :) Stay tuned!

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Matthew Taliaferro @jmtali.bsky.social · 11/06/2026

20/ Bottom line: these RNA localization elements are big, multipartite, and very complicated. We're digging further into the mechanisms behind their activity now. Stay tuned. Read the full preprint here 👇https://www.biorxiv.org/content/10.64898/2026.06.09.731215v1

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Matthew Taliaferro @jmtali.bsky.social · 11/06/2026

19/ There are a few possible reasons. Perhaps our in vitro structures may differ from those in cells, and/or perhaps important features (like G-quadruplexes) may be invisible to our methods.

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Matthew Taliaferro @jmtali.bsky.social · 11/06/2026

18/ With this pool, critical-element mutations stayed lethal, but we never saw mutations in a stem rescued by compensatory mutations. No clear evidence that these specific structures drive activity.

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Matthew Taliaferro @jmtali.bsky.social · 11/06/2026

17/ To test the functionality of those structures, we built another pool. We mutated every stem, plus made structure-preserving "compensatory" mutations that should restore the fold.

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Matthew Taliaferro @jmtali.bsky.social · 11/06/2026

16/ Elements this big made us wonder if structure matters. With Chase Weidmann, we solved the secondary structures of the 260 nt elements using SHAPE-MaP.

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Matthew Taliaferro @jmtali.bsky.social · 11/06/2026

15/ Again, shuffling the critical element killed activity. But the support elements tolerated even wide shuffling windows. So they work through bulk nucleotide content, with exact nucleotide order mattering much less.

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Matthew Taliaferro @jmtali.bsky.social · 11/06/2026

14/ Does the exact order of nucleotides matter, or just the overall content (how many adenosines, etc.)? To test this, we made thousands more variants where we kept the content of a window fixed but shuffled the order.

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Matthew Taliaferro @jmtali.bsky.social · 11/06/2026

13/ Same story. Any mutations in the critical element killed activity. The support element tolerated small mutation windows but not larger ones.

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Matthew Taliaferro @jmtali.bsky.social · 11/06/2026

12/ Could it be that deletions just throw off the spacing between key parts? To check, we left the length intact and instead slid a window of mutations across the element.

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Matthew Taliaferro @jmtali.bsky.social · 11/06/2026

11/ These critical elements aren't just a quirk of our workhorse neuronal cell line. With Michael Kiebler's lab, single-molecule imaging showed they're also essential for shipping RNA to axons in primary hippocampal neurons.

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Matthew Taliaferro @jmtali.bsky.social · 11/06/2026

10/ We also found "support elements". They tolerate small (~10 nt) deletions but not bigger (~50 nt) ones. So each localization element has at least two distinct functional regions.

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