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. 2024 May 30;12:RP90316. doi: 10.7554/eLife.90316

Figure 5. N- and C-terminal tRNA methyltransferase 1 (TRMT1) cleavage fragments exhibit alterations in RNA binding and tRNA modification activity.

(A) Schematic of wild-type TRMT1 and predicted TRMT1 fragments resulting from nonstructural protein 5 (Nsp5) cleavage at Q530N. (B) Immunoblot analysis of anti-FLAG purifications from human cells expressing vector control, full-length TRMT1, or TRMT1 cleavage fragments fused to the FLAG tag. The immunoblot was probed with anti-FLAG and anti-actin antibodies. (C) Nucleic acid stain of RNAs extracted from the indicated input or purified samples after denaturing PAGE. The migration pattern of 5.8 S rRNA (~150 nt), 5 S rRNA (~120 nt), and tRNAs (~70–80 nt) are denoted. (D) Immunoblot of TRMT1 expression in either control-wild-type (WT) or TRMT1-knockout (KO) human 293T cell lines. (E, F) Representative gel of primer extension assays to monitor the presence of dimethylguanosine (m2,2G) in tRNA-Met-CAU or mt-tRNA-Ile-GAU from the cell lines transfected with the indicated TRMT1 constructs. D, dihydrouridine; m1G, 1-methylguanosine; >, labeled oligonucleotide used for primer extension. Protein-RNA purification was repeated with comparable results (see source data for repeat).

Figure 5—source data 1. Raw uncropped immunoblots for Figure 5.

Figure 5.

Figure 5—figure supplement 1. Confocal microscopy images of 293T cells transiently transfected with constructs expressing tRNA methyltransferase 1 (TRMT1) or TRMT1 fragments fused with green fluorescent protein (GFP).

Figure 5—figure supplement 1.

Mitochondria were identified using mitochondrion-targeted red fluorescent protein (Mito-RFP) and nuclear DNA was stained with Hoechst. Overlap of red mitochondria and green GFP signal is displayed in the Merge panels.
Figure 5—figure supplement 1—source data 1. Raw uncropped microscopy images for Figure 5—figure supplement 1.