A tRNA-derived RNA Fragment Plays an Important Role in the Mechanism of Arsenite -induced Cellular Responses

Abstract

Chronic exposure to environmental heavy metals is a worldwide health concern. It is acknowledged to be an important cause of lower respiratory tract damage in children. However, the molecular mechanisms underlying the heavy metal-induced cellular stress/toxicity are not completely understood. Small non-coding RNAs (sncRNAs), such as microRNAs (miRNA) and more recently identified tRNA-derived RNA fragments (tRFs), are critical to the posttranscriptional control of genes. We used deep sequencing to investigate whether cellular sncRNA profiles are changed by environmental heavy metals. We found that the treatment of arsenite, an important groundwater heavy metal, leads to abundant production of tRFs, that are ~30 nucleotides (nts) long and most of which correspond to the 5'-end of mature tRNAs. It is unlikely for these tRFs to be random degradation by-products, as the type of induced tRFs is heavy metal-dependent. Three most inducible tRFs and their roles in arsenite-induced cellular responses were then investigated. We identified that p65, an important transcription factor belonging to NF-κB family and also a key factor controlling inflammatory gene expression, is a regulated target of a tRF derived from 5'-end of mature tRNA encoding AlaCGC (tRF5-AlaCGC). tRF5-AlaCGC activates p65, subsequently leading to enhanced secretion of IL-8 in arsenite response. In this study, we also identified that endonuclease Dicer and angiogenin temporally control the induction of tRF5-AlaCGC, providing an insight into the control of tRF biogenesis and subsequently the prevention of cellular damage.

Figure 1

Pipeline of analyses of Illumina high-throughput sequencing data. Flowchart of the sequencing data analyses is depicted.

Figure 2

Experimental validation of tRF-5s. (A) Sequence alignment of three validated tRF5s with their parental mature tRNAs and Northern probes. The letters in bold indicate the codon sequences. (B) Total RNA from A549 cells, treated with arsenite for 6 h at indicated concentrations was loaded to a denaturing polyacrylamide gel for Northern hybridization using probes indicated in panel A. Untreated cells were used as control. Total RNAs stained with ethidium bromide (EtBr) staining and Northern detection on the 5 S rRNA are 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 8 h, 1 day, and 5 days for the detection of tRF5-GluCTC, tRF5-ProTGG, and tRF5-AlaCGC, respectively. Data are representative of three independent experiments. (C) Densitometric analysis of the tRF bands from three Northern blots was performed for 1B, using the histogram function of Adobe Photoshop (San Jose, CA). Basically, the mean tRF intensity was normalized by the corresponding mean intensity 5 S rRNA and expressed as mean ± standard error (SE). For two bands of tRF5-GluCTC in the same treatment, their mean intensity was first calculated, followed by normalization. (D,E) The induction of tRF5-GluCTC, tRF5-ProTGG, and tRF5-AlaCGC were also inducible by arsentite in SAE cells. The Northern blot was done similarly as described in B, while the band intensity was quantified as described in C. Data shown are representative of three independent experiments. * and ** represent P < 0.05 and P < 0.01 respectively, relative to CN oligo-treated cells.

Figure 3

The role of tRFs in arsenite-induced inflammation. (A) A549 cells in 6-wells were transfected with 100 nM anti-sense oligos (anti-“tRF5-GluCTC”, -“tRF5-ProTGG”, -“tRF5-AlaCGC” or -“CN”). After 2 h post-transfection, cells were treated with 10 µM arsenite for 15 h. Untreated cells were used as controls. The nuclear fractions were prepared, followed by the SDS-PAGE gel running and Western blot, to detect the nuclear translocation of p65. (B) The supernatant of each group was harvested, and its IL-8 was measured by an ELISA kit from R&D (R&D system, Minneapolis, MN). Data shown are representative of three independent experiments. ^*P < 0.05 relative to CN oligo-treated cells.

Figure 4

The biogenesis of tRF5-AlaCGC is controlled by angiogenin and Dicer. (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 treated with arsenite for 6 or 15 h. Cells without the treatment were used as controls. Total RNAs were then subjected to Northern hybridization as described in Fig. 2. 5 S rRNA and EtBr staining were shown for equal loading. (B) The suppression of target proteins by each siRNA was confirmed by Real-time PCR. (C) The target specific suppression by siRNAs was also confirmed by Western blot after 40 h post transfection. (D) Densitometric analysis of the tRF bands from three Northern blots was performed for 4A, similarly as described in Fig. 2C. Data shown are representative of three independent experiments. * and ** represent P < 0.05 and P < 0.01 respectively, relative to CN oligo-treated cells at corresponding time point of arsenite treatment.