The Importance of AGO 1 and 4 in Post-Transcriptional Gene Regulatory Function of tRF5-GluCTC, an Respiratory Syncytial Virus-Induced tRNA-Derived RNA Fragment

Abstract

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants, the elderly, and immune-compromised patients. It is also a significant contributor to upper respiratory tract infection in the pediatric population. However, its disease mechanisms are still largely unknown. We have recently shown that a tRNA-derived RNA fragment (tRF) from the 5'-end of mature tRNA encoding GluCTC (tRF5-GluCTC), a recently discovered non-coding RNA, is functionally important for RSV replication and host gene regulation at the post-transcriptional level. However, how tRF5-GluCTC carries out the gene regulation is not fully known. In this study, we found that tRF5-GluCTC has impaired gene trans-silencing function in cells deficient of AGO1 or 4, while AGO2 and 3 seem not involved in tRF5-GluCTC-mediated gene regulation. By pulling down individual AGO protein, we discovered that tRF5-GluCTC is detectable only in the AGO4 complex, confirming the essential role of AGO4 in gene regulation and also suggesting that AGO1 contributes to the gene trans-silencing activity of tRF5-GluCTC in an atypical way. We also found that the P protein of RSV is associated with both AGO1 and 4 and AGO4 deficiency leads to reduced infectious viral particles. In summary, this study demonstrates the importance of AGO1 and 4 in mediating the gene trans-silencing function of tRF5-GluCTC.

Keywords: RSV; argonaute; post-transcriptional gene regulation; tRNA-derived RNA fragments.

Figure 1

AGO1 and 4 knockdown inhibit RSV-induced tRF5-GluCTC activity. (A) A549 cells in triplicate were transfected with 100 nmol/l of siRNA against human AGO1, 2, 3, or 4, or scrambled siRNA (Control) as a negative control in the presence of Pp-anti_GluCTC sensor and renilla (Rr) plasmids. At 6 h post-transfection, cells were mock- or RSV-infected at a multiplicity of infection (MOI) of 3 and then harvested at 15 h post-infection (p.i.) to measure luciferase activities. Pp luciferase values were normalized to Rr luciferase values. Data are representative of 3–4 independent experiments and expressed as means ± SE. * p < 0.05, relative to the paired white bar. (B) Cells were transfected with control or each AGO-specific siRNA as described in (A). At 24 h post-transfection, total RNA was prepared, followed by cDNA synthesis and qRT-PCR using each AGO-specific primer. AGO expression was normalized to that of 18S mRNA. Data are representative of three independent experiments and expressed as means ± SE.

Figure 2

AGO4 interacts with tRF5-GluCTC. (A) 293 cells expressing Flag-tagged AGO1, 2, 3, or 4 protein were mock-infected or infected with RSV, at MOI of 1, and total RNA was prepared, followed by Northern hybridization using the tRF5-GluCTC probe to detect the induction of tRF5-GluCTC by RSV infection. EtBr staining is shown for equal loading. EtBr, ethidium bromide. (B) Sample input was investigated by Western blot using either an anti-Flag antibody (AGO expression) or an anti-RSV antibody (viral protein expression). (C) Total lysates were prepared and followed by IP using an anti-Flag antibody. Flag-AGO bound RNA was purified and subjected to Northern blot for tRF5-GluCTC detection. The AGO expression in the pulldown complex was also investigated by Western blot to ensure the proper IP process. (D) To study the association of viral protein(s) with AGO complex, the membrane used in C was stripped and reprobed with an anti-RSV antibody. Data are representative of three independent experiments.

Figure 3

AGO4 knockdown reduces RSV replication. (A) A549 cells in triplicate were transfected with 100 nmol/L of siRNA against human AGO1, 2, 3, or 4, or scrambled siRNA (Control). At 24 h post-transfection, the cells were mock-infected or infected with RSV at MOI of 1 for 15 h and harvested for RSV titration assays. Data are representative of three independent experiments and expressed as means ± SE. * p < 0.05, relative to the control siRNA. (B) Cells, transfected with control or AGO4 siRNA, were infected with RSV and then harvested to prepare total cell lysates, followed by Western blot to check the expression of viral proteins using an anti-RSV antibody. β-actin was used as a control for equal loading of the samples. Data are representative of three independent experiments.

Figure 4

Targeting mechanism models of tRF5-GluCTC. (A) In the normal state, RSV-induced tRF5-GluCTC binds to AGO4, leading to an increase in the gene silencing activity. Upon AGO1 depletion, AGO1-associated sncRNAs are released and compete with RSV-induced tRF5-GluCTC for the loading onto AGO4, leading to less tRF5-GluCTC in AGO4 and consequently the reduced gene trans-silencing activity of tRF5-GluCTC. (B) Upon RSV infection, AGO1 binds to mRNA targets in a tRF5-GluCTC-independent manner, subsequently helping tRF5-GluCTC to find its targets. (C) Upon RSV infection, AGO1 or other unidentified molecular partners associate with mRNA targets and deliver them to the AGO4-tRF5-GluCTC complex.