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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

Thank you for developing LOV-BirA! Without it, this work wouldn't have been possible. It was wonderful to work together.

Alice Ting @aliceyting.bsky.social · Aug 27

Proximity-specific ribosome profiling using LOV-BirA reveals two distinct strategies for mitochondrially-localized translation: one for long coding sequences and one for short. It was a pleasure to contribute to this beautiful work from Jonathan Weissman @weissmanlab.bsky.social‬ and Jingchuan Luo

Reposted by Jingchuan Luo

Whitehead Institute @whiteheadinstitute.bsky.social · Aug 27

LOCL-TL allows researchers to study which proteins are being made, or translated, at specific locations within the cell. @jswlab.bsky.social developed this tool and used it to characterize translation happening at the mitochondria. wi.mit.edu/news/locally... @hhmi.org

Locally produced proteins help mitochondria function | Whitehead Institute

Jonathan Weissman and colleagues developed an optogenetic approach to study where in the cell proteins get made and used it to characterize two groups of proteins produced next to mitochondria. This c...

wi.mit.edu

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

See also the work from Shu-ou Shan's lab Zikun Zhu et al where they used an orthogonal approach to investigate cotranslational import into mitochondria, and similarly found that the process prioritizes large proteins with a bipartite signal.(cell.com/cell/fulltex...

cell.com

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(18) Looking forward, LOCL-TL provides a powerful tool to study localized translation across subcellular compartments under physiological conditions. There's so much more in the paper! Check out all the cool findings here. (cell.com/cell/fulltex...

cell.com

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(17) This pathway prioritizes long proteins of prokaryotic origin, ensuring their proper expression and import into the mitochondrial matrix. The localized translation of short proteins could be a novel way to regulate OXPHOS and may have co-evolved with the mitochondrial genome.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(16) In summary, a majority of mitochondrial proteins are imported post-translationally. A subset uses localized translation. Long proteins are cotranslationally targeted by a bipartite signal. Short proteins, however, use translation-independent mRNA targeting mediated by AKAP1.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(15) Localized translation of long proteins is conserved in yeast, unlike that of short proteins. As these proteins often originate from prokaryotes, this suggests that localized translation is an ancient mechanism that ensures their proper synthesis and mitochondrial import.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(14) In AKAP1 null mutants, oxidative phosphorylation (OXPHOS) components are significantly reduced, suggesting that localized translation may represent a novel regulatory mechanism for OXPHOS, as electron transport chain (ETC) subunits are highly enriched in this pathway.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(13) AKAP1’s ability to recruit mRNAs correlates with its ability to mediate local translation, revealed by RIP-qPCR experiments with different AKAP1 variants and short CDS mutant reporters, supporting the idea that AKAP1 directly interacts with RNA to recruit the short CDS.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(12) We found that the mitochondrial outer membrane protein AKAP1 mediates the recruitment of short CDS transcripts through its KH domain and Tudor domains, but not its PKA-helix.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(11) However, local recruitment of short CDS transcripts (e.g. NDUFB9) is uncoupled from protein import, which requires a functional MTS.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(10) Localized recruitment of short proteins is mediated by RNA, independent of the MTS and protein translation. A synthetic reporter with the UTRs from the COX7A2 gene (one of the top hits), a single intron, and a partial mCherry sequence was sufficient for recruitment.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(9) We found that cells prevent the cotranslational import of most mitochondrial proteins with an inhibitory signal. In contrast, long proteins are recruited cotranslationally by a bipartite signal: an MTS and a downstream region (100-350aa) that lacks the inhibitory signal.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(8) There are distinct mechanisms for each group. Long proteins initiate synthesis in the cytosol, and through co-translational recruitment, finish at the mitochondria. In contrast, short protein mRNAs are recruited to the mitochondria before translation begins.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(7) We found that ~20% of nuclear-encoded mitochondrial proteins are translated locally at mitochondria. These fall into two groups by length: long proteins (>400aa), enriched in matrix metabolic enzymes, and short proteins (<200aa), enriched in electron transport chain subunits.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(6) We next applied LOCL-TL to identify genes translated at the mitochondria. Our method demonstrated high specificity, as 99% of our positive mitochondrial transcripts were also found in the mito APEX-seq dataset (sciencedirect.com/science/arti...), which labels mRNA near mitochondria.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(5) We benchmarked LOCL-TL in the ER system, where it robustly enriched for secretory proteins known to be translated locally at the ER membrane.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(4) To solve this, we collaborated with @aliceyting.bsky.social Song-Yi Lee to develop an optogenetic tool, the LOV-domain Controlled Ligase for Translation Localization (LOCL-TL), to label and pull down locally translated ribosomes for ribosome profiling under normal growth conditions.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(3) A decade ago, our lab developed proximity-specific ribosome profiling (science.org/doi/10.1126/...) to quantitatively monitor localized translation. However, this method requires cells to grow in biotin-depleted media, which causes a strong mitochondrial and cell-growth defect.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(2) For example, are some proteins synthesized near mitochondria? If so, which ones? How is this process regulated and why is it important? Addressing these questions can reveal crucial insights into protein import, mito-nuclear coordination, and mitochondrial homeostasis.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(1) Localized translation enables the spatial and temporal regulation of protein expression, which is crucial for synaptic plasticity, cell migration, and organelle homeostasis. While well-studied at the ER and neuronal synapses, other systems in mammals remain poorly understood.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

Thanks to all fantastic coauthors Stuti Khandwala Jingjie(JJ)Hu @zebulonlevine.bsky.social. Thanks to all wonderful collaborators @aliceyting.bsky.social Song-Yi Lee who engineered LOV-BirA @harperlabhms.bsky.social Kelsey Hickey for the proteomic studies

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

I'm thrilled to share our work from @weissmanlab.bsky.social (www.sciencedirect.com/science/arti...). We developed LOCL-TL, an optogenetic approach for monitoring localized translation in mammalian cells. LOCL-TL revealed two distinct strategies for mitochondrially localized translation.

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Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(17) This pathway prioritizes long proteins of prokaryotic origin, ensuring their proper expression and import into the mitochondrial matrix. The localized translation of short proteins could be a novel way to regulate OXPHOS and may have co-evolved with the mitochondrial genome.

Jingchuan Luo @jingchuanluo.bsky.social · Aug 27

(16) In summary, a majority of mitochondrial proteins are imported post-translationally. A subset uses localized translation. Long proteins are cotranslationally targeted by a bipartite signal. Short proteins, however, use translation-independent mRNA targeting mediated by AKAP1.

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