Combined w. work from Jackie Trotter & Doug Fields Labs, we propose a RNA switch model:

- During transport, hnRNP-F binds unstructured MLS to inhibit translation.

- At the cell periphery, membrane-bound Fyn kinase phosphorylates & releases hnRNP-F to initiate translation.

7/8

Next, we used mass spec to ask what proteins associate with the MLS mRNA region.

We found hnRNP-F and proteins involved in translation!

Our structure of the MLS also had the canonical hnRNP-F-binding GGG(A) motifs!

6/8

Importantly, the MLS can be used to deliver CDS cargos throughout oligodendrocyte processes.

Here, we used EGFP as a reporter cargo.

5/8

We found a ~120-nt region sufficient for transport that we call the MLS.

Comparing to decades-old results from the Carson lab:
- Their RTS regions was not validated.
- But our MLS partially overlaps with their RLR region.

4/8

We asked which part of the ~1.5-kb Mbp 3' UTR is important for mRNA transport?

Ved applied 2 orthogonal techniques to primary olives:
- DMS-MaPseq to solve the RNA structure
- SLAP-seq - a method he innovated combining ~270 reporters and Boyden chambers cultures

3/8

This is the creepiest Halloween decoration I’ve ever seen… IRL it’s very lifelike 😱

# BerkeleyLife

Many distal ends are enriched in GFAP.

We don't know what this means yet - maybe these can become endfeet that contact blood vessels. Astrocyte processes are heterogeneous in vivo (some contact blood vessels, others contact synapses) - like axons vs. dendrites in neurons.

7/x

By cryo-ET, we could see a lot of intermediate filaments (IFs). The ratio of IFs:microtubules was very high ~7:1.

Image below shows: IFs (blue), microtubules (green), actin (yellow).

6/x

Microtubules in primary processes are highly stable as indicated by:
- Acetylation
- Detyrosination
- MIPs (microtubule inner proteins)
- IF content

5/x

And we confirmed this with cryo-ET.

4/x

We used live-cell imaging with plus-end binding protein EB3 to determine microtubule polarity in astrocytes for the first time. They are ~88% plus-ends out.

3/x

We isolated astrocytes using the immunopanning method. Most images are from cells grown in serum-free media for ~2 weeks... they make beautiful processes!

Microtubules & GFAP dominate in thick long primary processes.

And we see a novel actin structure - reticular webbing!

2/x

Saw a family of quails! How many do you see?

Saw some cool mushrooms on campus today! Near Euclid and Hearst.

en.m.wikipedia.org/wiki/Clathru...

In these trying times, grateful for little kindnesses 🥰

Spotted on my walk home!

#BerkeleyLife

Jennifer Doudna: I would not be here if not for NIH training grants!

UC Berkeley @standupforscience.bsky.social

Tough times call for nerd humor!

Just taught action potentials this week…