Identification of two novel functional tRNA-derived fragments induced in response to respiratory syncytial virus infection

Abstract

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection (LRTI) in children from infancy up to early childhood. Recently, we demonstrated that RSV infection alters cellular small non-coding RNA (sncRNA) expression, most notably the tRNA-derived RNA fragments (tRFs). However, the functions of the tRFs in virus-host interaction are largely unknown. Herein, we examined the role of three RSV-induced tRFs derived from the 5-end of mature tRNAs decoding GlyCCC, LysCTT and CysGCA (named tRF5-GlyCCC, tRF5-LysCTT and tRF5-CysGCA, respectively) in controlling RSV replication. We found that tRF5-GlyCCC and tRF5-LysCTT, but not tRF5-CysGCA, promote RSV replication, demonstrating the functional specificity of tRFs. The associated molecular mechanisms underlying the functions of tRF5-GlyCCC and tRF5-LysCTT were also investigated. Regulating the expression and/or activity of these tRFs may provide new insights into preventive and therapeutic strategies for RSV infection. The study also accumulated data for future development of a tRF targeting algorithm.

Fig. 1.

Experimental validation of tRF-5s. (a) Sequence alignment of tRF-5s with their parental mature tRNAs and Northern probes. The length of each sequence and CCA, post-transcriptionally added to the 3′-end of tRNA, are indicated in parentheses and brackets, respectively. (b) Total RNA from indicated treatments in A549 cells was loaded to a denaturing polyacrylamide gel for Northern hybridization using probes indicated in panel A. Total RNA is shown for equal loading. The positions of tRF-5 and mature tRNA are indicated on the right; molecular size markers are indicated on the left. The blot was exposed for between 8 and 48 h. Data are representative of two–three independent experiments. (c) Densitometric analysis of the tRF bands was performed for Fig. 1(b), using the histogram function of Adobe Photoshop. Basically, the mean tRF intensity was normalized by the corresponding mean intensity of total RNA.

Fig. 2.

Effects of tRFs on RSV replication. (a, b) 100 nM anti-sense oligos (‘anti-tRF5-GlyCCC’, ‘anti-tRF5-LysCTT’, ‘anti-tRF5-CysCGA’ or ‘anti-CN’) were transfected into A549 cells. Two hours post-transfection, cells were mock- or RSV-infected, followed by washing using FK-12 with 2 % FBS for three times and total virus harvesting at 15 h p.i. (hours post-infection) Viral infectious particles were quantified by immunostaining (a). Viral protein synthesis was investigated by Western blot (b). (c, d) A549 cells were transfected with 100 nM of indicated mimic oligos. Two hours post-transfection, cells were mock- or RSV-infected. Two hours post-infection, the supernatant was removed and cells were incubated for additional 15 h, viral titration (c) and viral protein assays (d) were determined. (a, c) Values at the y-axis are a representative of three to four independent experiments and are expressed as mean±standard error (se). *On bars as indicated in the panel denotes a P-value <0.05, relative to the first plain bar [RSV-infected and CN for antisense oligo (α-CN shown in a) or mimic oligo (CN shown in c)]. (e, f) The histogram function of Adobe Photoshop was used to quantify viral bands in (b, d). The intensity sum of viral proteins was normalized by the corresponding β-actin intensity.

Fig. 3.

Gene trans-silencing function of tRF5-GlyCCC. (a) The sensor plasmid ‘Pp-anti_GlyCCC’ was constructed by inserting an oligonucleotide, which was complementary to tRF5-GlyCCC, into EcoRI/XhoI sites of pcDNA3.1-Zeo(+)-Pp, as described previously [41]. An empty pcDNA3.1-Zeo (+)-Pp vector was used as a control (designated as ‘Pp-control’). To dissect the target interaction regions of tRF5-GlyCCC, the mutated oligonucleotides were inserted into luciferase reported as instructed. (b) To investigate the effect of RSV-induced tRF5-GlyCCC on mRNA expression, A549 cells were co-transfected with Pp-anti_GlyCCC sensor plasmids (firefly plasmids), pRL-CMV plasmids expressing Rr (renilla luciferase) and anti-tRF5-GlyCCC oligonucleotides (anti-GlyCCC), using Lipofetamine 2000 according to the manufacturer’s instructions (Invitrogen). Pp-control plasmids and anti-control oligonucleotides were used as negative controls. After 2 h of transfection, the cells were infected with mock or RSV at an m.o.i. of 1. At 15 h p.i., cells were lysed for assays by using a dual-luciferase kit (Promega, Madison, WI). Data normalization was done by correcting Pp luciferase by the Rr luciferase within the same group and normalized value in the RSV-infected group was further normalized to its corresponding mock-infected group within same oligo treatment. Values at the y-axis are a representative of three to four independent experiments and are expressed as mean±standard error (se). ** On second black bars (RSV infected and Pp-anti_GlyCCC and anti-control oligo-treated) as indicated denotes a P-value<0.01, relative to the second plain bar (mock infected and anti-control for antisense oligo). (c) To identify targeted regions of tRF5-GlyCCC, A549 cells were co-transfected with Pp-anti_GlyCCC sensor plasmids containing various mutation in regions as indicated and Rr expressing plasmids. Pp-anti_GlyCCC sensor (Pp-WT) and Pp-vector (Pp-CN) plasmids were used as positive and negative controls, respectively. The infection and luciferase normalization were done as described in (b). Values at the y-axis are a representative of three to four independent experiments and are expressed as mean±standard error (se). ** On black bars, as indicated, denotes a P-value<0.01, relative to the corresponding open or plain bar in the same group. (d) The gene trans-silencing function of tRF5-GlyCCC was also done in the context of tRF5-GlyCCC-mimic treatment. For ectopic expression of tRF5-GlyCCC, tRF5-GluCCC-mimic in siRNA format was designed and synthesized from Ambion. The same design with a scrambled sequence was used as a control (‘CN Oligo’). A549 cells were cotransfected with mimics (tRF5-GlyCCC specific or CN oligo), Pp plasmids (Pp-anti_GlyCCC WT or containing mutations in the middle and 5′-end of tRF5-GlyCCC) and Rr-expressing plasmids using Lipofectamine 2000. Cells were harvested for dual-luciferase assays at 30 h post-transfection. Data normalization was done by correcting Pp luciferase by the Rr luciferase within the same group and the normalized value of the tRF5-GlyCCC mimic-treated group was further normalized to its corresponding CN-oligo-treated group within the same Pp vector. Values at the y-axis are a representative of three to four independent experiments and are expressed as mean±standard error (se). ** On first black bars (Pp-WT transfected and tRF5-GlyCCC mimic-treated), as indicated, denotes a P-value<0.01, relative to the first open or plain bar (Pp-WT transfected and CN mimic-treated).

Fig. 4.

Gene trans-silencing function of tRF5-LysCTT. (a) To identify whether tRF5-LysCTT has the gene trans-silencing function and associated regulatory mechanism, the sensor plasmid ‘Pp-anti_LysCTT’ or its mutants were constructed as described in Fig. 3(a). (b). To define the gene trans-silencing function of tRF5-LysCTT, dual-luciferase assays were done as described in Fig. 3(b), except Pp-anti_LysCTT sensor plasmids (firefly plasmids) and anti-LysCTT oligonucleotides were used. ** On second black bars (RSV infected, Pp-anti_LysCTT and anti-control oligo-treated), as indicated, denotes a P-value<0.01, relative to the second open or plain bar (mock-infected and Pp-anti_LysCTT and anti-control oligo-treated). (c) To identify targeting mechanisms of tRF5-LysCTT, the experiment was carried out as described in Fig. 3(c) using the plasmids designed in Fig. 4(a). (d) To confirm the interacting regions identified in Fig. 4(c), tRF5-LysCTT mimic was used to investigate its suppression on the luciferase expression of Pp-anti-LysCTT (Pp-WT) and motif mutants identified in Fig. 4(c).

Fig. 5.

Effects of tRF5-GlyCCC and tRF5-LysCTT on RSV-induced cytokines and chemokines. A549 cells were transfected with 100 nM of indicated anti-oligos (a) or tRF5 mimic (b). Two hours post-transfection, cells were mock- or RSV-infected. Fifteen hours post-infection, supernatant was harvested to measure secretion of cytokines/chemokines by Bioplex or ELISA, as described previously [42]. ELISA kits for IL-8/RANTES and IFN-β were from R and D (R and D system, Minneapolis, MN) and PBL (PBL Interferon Source, Piscataway, NJ), respectively. Data shown are representative of three independent experiments. * P<0.05 relative to CN-treated and RSV-infected cells (black bars).

Fig. 6.

tRF5-GlyCCC and tRF5-LysCTT are generated by Angiogenin. (a) A549 cells were transfected with 100 nM of siRNA against indicated proteins or scrambled siRNA as a negative control. At 40 h post-transfection, the cells were mock- or RSV-infected for 15 h. Total RNAs were then subjected to Northern hybridization as described in Fig. 1(b) (top panel). EtBr staining is shown for equal loading (bottom panel). (b) The suppression of target proteins by each siRNA was confirmed by Western blot.