ELAC2, an Enzyme for tRNA Maturation, Plays a Role in the Cleavage of a Mature tRNA to Produce a tRNA-Derived RNA Fragment During Respiratory Syncytial Virus Infection

Figures

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  Figure 1. The effect of tRF5-GlnCTG on RSV replication. A549 cells in duplicate were transfected with 120 nmol/l of an antisense oligo against tRF5-GlnCTG (α-GlnCTG) or scrambled oligo (α-Control) using Lipofectamine 2000. At 2 h post-transfection, the cells were mock-infected or infected with RSV at an MOI of 1 for 15 h. (A) Total viruses were harvested and infectious particles were quantified by immunostaining using an anti-RSV antibody. (B) Total proteins were prepared, and the expression of viral proteins was measured by Western blot using an anti-RSV antibody. RSV proteins are indicated on the right and molecular size markers are on the left. β-actin was used as a control for equal loading of the samples. The sequence of α-GlnCTG and α-Control is shown in Supplementary Table S1. Data are representative of three independent experiments. *P < 0.05, relative to the white bar.

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  Figure 2. Experimental validation and characterization of tRF5-GlnCTG. (A) Sequence alignment of tRF5-GlnCTG with its parental mature tRNA and Northern probe (upper panel), and the illustration of primers and the 3’ RNA linker for tRF detection by qRT-PCR (lower panel). (B) A549 cells were mock- or RSV-infected at an MOI of 1. Cytoplasmic (Cyto) and nuclear (Nu) RNAs were purified at 24 h p.i. (left panel) or Cyto RNAs were prepared at 6 and 24 h p.i. (right panel), followed by Northern hybridization to detect tRF5-GlnCTG using the probe indicated in (A). Ethidium bromide (EtBr) staining is shown for equal loading. The size of tRF5 and mature tRNA is indicated on the right; molecular size markers are indicated on the left. Data are representative of 2–3 independent experiments. (C) SAE cells were mock- or RSV-infected at an MOI of 1 for 15 h, followed by Cyto and Nu RNA preparation and then Northern hybridization to detect tRF5-GlnCTG using the probe indicated in (A). EtBr staining is shown for equal loading. Data are representative of three independent experiments. (D) A549 cells in triplicate were mock-infected or infected with RSV at an MOI of 1 for 24 h, followed by Cyto RNA…

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  Figure 3. ELAC2 is responsible for the induction of tRF5-GlnCTG. (A) A549 cells were transfected with 100 nmol/l of siRNA against individual indicated nuclease or control siRNA. At 40 h post-transfection, the cells were mock- or RSV-infected at an MOI of 1 for 24 h, followed by Cyto RNA preparation and Northern hybridization for the detection of tRF5-GlnCTG. The individual gene knockdown was verified by qRT-PCR (down panel). (B) Cells were transfected with siRNAs and then mock- or RSV-infected as described in (A), followed by total protein preparation and then Western blot using antibodies against individual targets. β-actin was used as a loading control. (C) Compartmental locations of ELAC2. Cells were uninfected or infected with RSV at an MOI of 1 for 24 h, followed by subcellular fractionation preparation for mitochondria-free cytoplasm (Mito-free Cyto), nuclei (Nu), and mitochondria (Mito) and then Western blot using the indicated antibodies. The purity of fractions was assessed by compartment-specific proteins. The succinate dehydrogenase complex subunit A (SDHA), Lamin B1, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serve as a mitochondrial, nuclear, and mitochondria-free…

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  Figure 4. The contribution of RSV proteins to the induction of tRF5-GlnCTG. (A) A549 cells in triplicate were transfected with plasmids encoding V5-tagged RSV N, P, NS1, NS2, or empty vector (CN, negative control for viral protein expression). Vectors of Pp-anti_GlnCTG_WT (containing the target sequence of tRF5-GlnCTG) or Pp-anti_Control (without tRF5-GlnCTG target sequence) were also co-transfected with renilla luciferase plasmids (Rr, luciferase expression internal control). At 30 h post-transfection, the cells were harvested for the dual-luciferase assay. Pp luciferase values were normalized to their corresponding Rr luciferase values and then the normalized value of Pp-anti_GlnCTG_WT was normalized to that of Pp-anti_Control vector. Data are representative of three independent experiments. **P < 0.01, relative to the first bar. (B) 293 cells were transfected with individual V5-tagged viral proteins as indicated. CN and P-expressing plasmids were used as controls. After 30 h, the cells were harvested and immunoprecipitation was done using the anti-V5 antibody, followed by Western blot using the anti-ELAC2 and anti-V5 antibodies. Proper expression of ELAC2 and V5-tagged RSV proteins was…