Journal: bioRxiv

Article Title: Active transport of tRNAs facilitates distributed protein synthesis

doi: 10.64898/2026.01.26.698744

Figure Lengend Snippet: (A) Representative images of endogenous pan tRNAs (pink) and quantification of perinuclear enrichment relative to cytoplasmic levels in ARVMs infected with an adenovirus encoding short hairpins to knockdown Kif5b (Ad-shKif5b, light blue bar) or a scramble (Ad-ShCtrl, light grey bar) control sequence for 96h. (B) Representative Cy5-tRNA tracks (color coded by maximum distance traveled, dark blue, <1 µm; dark red ≥ 5 µm) and representative tRNA kymograph (arrow points to specific track) in myotubes after 72h treatment with 50 nM siRNAs targeting scramble sequences (siScr, grey bar) or siRNAs targeting KIF5b (si Kif5b , light blue). Frequency distribution of maximum distance travelled per tRNA punctum and measurements of the percentage of tracks ≥1.5 microns ( n = 5 myotubes, with approximately 500 puncta/video analyzed). (C) Representative image of endogenous Leucyl-tRNA synthetase (LeuRS, blue) and microtubules (grey) in an ARVM, with yellow arrows indicating the area of zoom where LeuRS is decorating microtubules. (D) Representative images of LeuRS (blue) localization in ARVMs treated overnight with DMSO (grey bars) or 500nM nocodazole (Noc, red bars) and quantification of perinuclear enrichment relative to cytoplasmic levels. (E) Representative images of pan tRNA (pink) and microtubule in ARVMs treated 96h with an adenovirus encoding short hairpins targeting a scramble sequence (Ad-sh-Scr, grey) or LeuRS (Ad-shLRS, blue) and quantification of the percentage of tRNA puncta colocalizing with microtubule tracks. (F) Representative images of ARVMs transduced with Ad-shScr (shScr) or Ad-shLeuRS (shLRS) after 96h after infection and staining of LamTOR4+ ELVs (green) and pan-tRNA (pink) and quantification of the percent of ELVs colocalized with tRNA puncta. (G) Representative images of Cy5-tRNA tracks (color coded by maximum distance traveled, dark blue, <1 µm; dark red ≥ 5 µm) and representative kymographs (tRNA track, pink) from the selected tRNA puncta (arrow) in C2C12s treated for 96h with 50nM siRNAs to knockdown LeuRS (siLRS, blue) or a scramble sequence (siScr, grey) and frequency distribution of maximum distance travelled by puncta, and measurements of the percentage of tracks ≥1.5 microns ( n = 9 myotubes, with approximately 500 puncta/video analyzed). (H) Schematic of how the BC-LI-0186 (LeuRS-RagD inhibitor, LRI), which inhibits LeuRS from binding the RagD site on the outside of endolysosomes (ELVs), functions. (I) Representative images of LeuRS (purple) and ELV (green) staining in ARVMs treated with DMSO or 10 µM LRI for 24 hours (yellow arrows indicate sites of LeuRS-ELV colocalization) and quantification of the abundance of LeuRS-ELV colocalization puncta. (J) Representative images of ARVMs treated for 48h with DMSO or 10 µM LRI and stained for endogenous pan tRNAs (pink), microtubules (silver) and ELVs (green) with yellow box indicating area of zoom, and yellow arrows indicate ELVs with tRNAs on the outside. Quantifications of the percent of ELVs colocalized with tRNA. All experiments were done in biological triplicate ( N = 3), with 10 cells imaged per biological replicate ( n = 10), unless noted. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as determined by two-sided Student’s t-test.

Article Snippet: In vitro pharmaceuticals : Nocodazole (500 nM in DMSO, Thermo Fisher), Puromycin (2uM, A Sigma-Aldrich), Blebbistatin (5 μM in DMSO, Cayman Chemical), Pol3i inhibitor CAS 577784-91-9 (10uM, CAS 577784-91-9, Sigma Aldrich), Lars-RagD inhibitor BC-LI-0186 (10uM, LRI, MedChem Express).

Techniques: Infection, Knockdown, Control, Sequencing, Transduction, Staining, Binding Assay