We hope RiboExM and ALIBi will come in handy for others out there studying subcellular processes. Thanks to our labmates and collaborators, and our editors and reviewers at Science for helping us improve this work over many iterations! (end)

Bonus: we also collected data on ER-localized mRNA splicing isoforms, and on proteins associated w/ nuclear and nucleolar pre-ribosomes. Might be worth investigating as potential ribosome biogenesis factors. Outside the scope of this work, but hopefully useful to someone! (17/18)

Finally, Zijian applied RiboExM to hESC-iNeurons. Others have shown that most translation in distal neurites is done by monosomes. But upon treatment w/ BDNF and ACEA, which enhance local translation in distal neurites, RiboExM picked up increased abundance of polysomes. (16/18)

…Zijian expressed tagged versions of mRNAs encoding Mut and Pcc (proteins involved in B12 utilization in the mitochondrial matrix). RiboExM revealed that they preferentially associate w/ Rps25-lacking OMM ribosomes. Imaging specialized ribosomes in situ–that’s a first! (15/18)

By tagging Rps25 and a stoichiometric 40S protein, Zijian used RiboExM to image these “specialized” Rps25-lacking ribosomes at the OMM! And because our lab had previously shown differential binding of Rps25-lacking ribosomes to certain mRNAs… (14/18)

Next, we used ALIBi to capture ribosomes at the outer mitochondrial membrane (OMM), and found that some ribosomal proteins were depleted in OMM ribosomes. One of these is Rps25, which the Barna lab had previously found to be substoichiometric (not all ribosomes have it). (13/18)

(Thanks to Josh Black for telling me about the Vapa paper at #cshltranscon !) (12/18)

Is Lsg1 moonlighting as a translational regulator at the ER? How? We’re not sure, but Sutjita et al. (PMID 39133101) recently showed that a protein called Vapa tethers Lsg1 to the ER. Whether tethered Lsg1 still binds 60S, and whether that affects ER translation, is TBD🤔(11/18)

When we knocked down Lsg1 and used ALIBi to capture ER ribosome-associated mRNAs, we were intrigued to see that the association of specific mRNAs with ER ribosomes was disproportionately affected–for example, Vamp3 and Robo4, whose protein levels also change accordingly. (10/18)

Sec61a1 and Ufm1 made sense–they’re known components of the ER translocon and ER ribosome quality control, respectively. But Lsg1, Nmd3, Znf622, and eIF6 are ribosome biogenesis factors that mediate the final maturation of 60S in the cytosol…what are they doing at the ER? (9/18)

Using ALIBi to affinity purify ER ribosomes for TMT-MS, we found that 60S ribosomal proteins were relatively abundant, which corroborated Zijian’s result. A few other proteins were also enriched in ER translational machinery compared to cytosolic translational machinery… (8/18)

Oddly enough, I’d just come to a similar conclusion using a tool that I’d developed with @rna-bound.bsky.social and Kelsie Wysong: ALIBi, an optogenetically activated split “BirA” biotin ligase that enabled us to proximity biotinylate ribosomes at an organelle of interest. (7/18)

RiboExM can be combined with markers for organelles and can capture a panorama of ribosomes throughout an entire cell. That’s how Zijian noticed that 60S subunits were more abundant than 40S at the endoplasmic reticulum… (6/18)

Across several benchmarking experiments, Zijian’s image processing pipeline correctly identified individual free subunits, monosomes, and polysomes based on the size, intensity, and colocalization of immunofluorescent signals coming from the tagged 40S and 60S proteins. (5/18)

In pursuit of this question, my labmate Zijian Zhang with Yunhao Bai developed RiboExM, an expansion microscopy technique using tagged ribosomal proteins to visualize single 40S and 60S ribosome subunits at ~25 nm effective resolution–on a confocal 🔬! (4/18)

It’s known that specific mRNAs localize to specific subcellular compartments, seemingly so that the encoded protein is already where it needs to be once translated. But what about the translational machinery itself–is it different in different parts of a cell, too? (3/18)

This started out as two separate projects, actually! 🤝 @mbarnalab.bsky.social has long been interested in comparing the composition of ribosomes in different parts of a cell, since localized mRNA translation is such an important aspect of gene regulation and protein homeostasis. (2/18)

Belated post! If you haven’t seen our recent work www.science.org/doi/10.1126/... , here’s a 🧵 highlighting the two techniques we developed to study subcellular populations of mammalian ribosomes and some cool findings these tools helped us uncover: (1/18)