Informal Seminar - Hirotaka Toh & Kotaro Tomuro

○Kotaro Tomuro1,2, Yuichi Shichino1`, Shintaro Iwasaki1,2

(1 RIKEN Cluster for Pioneering Research, 2 Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo)

Keywords: Local translation, Ribosome profiling, Translation regulation

Local translation at specific subcellular regions including membranous and membrane-less compartments has been suggested to define appropriate destinations of proteins and ultimately maintain cellular homeostasis. Despite its importance, a comprehensive landscape of local translation is still far from complete due to a lack of simple and versatile analysis tools. Here, we developed a method termed "APEX-Ribo-Seq" that integrates ribosome profiling (Ribo-Seq) with APEX2-based proximity labeling and mapped the local translation status across 14 subcellular regions (ER membrane, mitochondrial outer membrane, peroxisome, cis-Golgi, trans-Golgi, lysosome, endosome, early endosome, centrosome, plasma membrane, cell junction, intermediate filaments, nuclear pore, and clathrin-coated structures). Comparative analysis revealed an atlas of local translation ~ 3000 genes, kinetic features associated with co-translational membrane insertion of nascent chains, and responsible RNA-binding proteins. We also examined local translation that is regulated by the cell cycle at centrosomes, by stress in stress granules and autophagosomes, and in distant locations such as synapses, dendrites, and axons in neurons. Overall, our strategy and datasets provide a landscape of the spatial translatome at subcellular resolution and its regulation in response to external and internal cues.

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○Hirotaka Toh1, Tomoya Fujita1,2, Takeshi Yokoyama3,4, Hideki Taguchi2,5,

Takuhiro Ito6, and Shintaro Iwasaki1,7

(1RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering

Research,2School of Life Science and Technology, Tokyo Institute of Technology,

3Laboratory for Protein Functional and Structural Biology, RIKEN Center for

Biosystems Dynamics Research, 4Graduate School of Life Sciences, Tohoku

University, 5Cell Biology Center, Institute of Innovative Research, Tokyo Institute

of Technology, 6Laboratory for Translation Structural Biology, RIKEN Center for

Biosystems Dynamics Research, 7Department of Computational Biology and

Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo)

Keywords: Ribosome profiling, Ribosome rotation, S1, Conformational ramp

Ribosome profiling — RNase footprinting of ribosome-bound mRNA — has been

a unique and powerful method, applied to widespread organisms to survey

ribosome traversal along mRNAs. In contrast to eukaryotes, bacterial ribosome

footprints show a broad range of sizes, reflecting the differential states of ribosomes.

However, the origin remains unclear. Here, we show that the rotated state of

ribosomes extends bacterial ribosome footprints at the 5′ ends. Exploring a series

of translation elongation inhibitors, bacterial ribosome profiling demonstrated that

the rotated state of ribosomes results in long footprints. Along the subunit rotation,

ribosomal protein S1 — a 30S-subunit RNA-binding protein — sterically protects

mRNA at the 5′ ends of the ribosome from RNase digestion. Combining these

findings, we established an improved method for bacterial ribosome profiling with

translation inhibitor cocktails, which can monitor ribosome subunit rotation. We

found that the subunit rotation was highly retarded at the 5-13 codons of ORF. Our

results provide an approach to probe the conformational diversity of ribosomes in

bacterial transcriptomes.