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

Abstract

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in young children. However, effective treatment against RSV is unavailable. tRNA-derived RNA fragments (tRFs) are a recently discovered family of non-coding RNAs. We made an early observation that RSV infection causes significant induction of tRFs, which are mainly derived from the 5'-end of mature tRNAs (tRF5). However, their functions and biogenesis mechanism are not fully understood. Herein, we identified an enzyme responsible for the induction of a functional tRF5 derived from tRNA-Gln-CTG (tRF5-GlnCTG). We found that tRF5-GlnCTG promotes RSV replication and its induction, assessed by Northern blot and a new qRT-PCR-based method, is regulated by ribonuclease ELAC2. ELAC2-mediated tRF5 induction has never been reported. We also found that ELAC2 is associated with RSV N and NS1 proteins. Given the fact that tRF5-GlnCTG plays a role in RSV replication, the identification of ELAC2 being responsible for tRF5-GlnCTG induction could provide new insights into therapeutic strategy development against RSV infection.

Keywords: ELAC2; RSV; biogenesis; tRF; viral replication.

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.

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 preparation and then qRT-PCR for the detection of tRF5-GlnCTG. The tRF5-GlnCTG value was normalized to that of RNU6. The sequence of forward and reverse primers are indicated in (A) and Supplementary Table S1. Data are representative of three independent experiments. **P < 0.01, relative to the white bar. (E) Left panel: Sequence alignment of tRF5-GlnCTG with a sensor plasmid containing targeting sequence of tRF5-GlnCTG (Pp-anti_GlnCTG_WT) and three mutant luciferase sensor plasmids (Mut3, MutM, and Mut5). Mutated nts are in red. Right panel: A549 cells in hexaplicate were co-transfected with indicated Pp luciferase sensor plasmids and internal control renilla luciferase plasmids (Rr). A sensor plasmid lacking the target site (Pp-anti_Control vector or CN) was used as a negative control. At 6 h post-transfection, cells were mock- or RSV-infected at an MOI of 1 and then harvested at 24 h p.i. to measure luciferase activities. The relative luciferase activity (Pp/Rr values) was calculated. Data are representative of three independent experiments. *P < 0.05, relative to the paired white bar.

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 cytoplasmic marker, respectively. Data are representative of 2-3 independent experiments. (D) RNA was prepared from subcellular fractionated compartments as described in (C). The same amount of 10 µg in all fractionated RNAs was loaded to a denaturing polyacrylamide gel for Northern hybridization to detect tRF5-GlnCTG. The loading ratio of each fraction was: all Mito RNAs vs 1/25 for Nu RNA vs 1/100 for Mito-free Cyto RNA. Data are representative of 2-3 independent experiments.

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 confirmed in sample input. Data are representative of three independent experiments.