Transfer RNA demethylase ALKBH3 promotes cancer progression via induction of tRNA-derived small RNAs

Abstract

Transfer RNA is heavily modified and plays a central role in protein synthesis and cellular functions. Here we demonstrate that ALKBH3 is a 1-methyladenosine (m1A) and 3-methylcytidine (m3C) demethylase of tRNA. ALKBH3 can promote cancer cell proliferation, migration and invasion. In vivo study confirms the regulation effects of ALKBH3 on growth of tumor xenograft. The m1A demethylated tRNA is more sensitive to angiogenin (ANG) cleavage, followed by generating tRNA-derived small RNAs (tDRs) around the anticodon regions. tDRs are conserved among species, which strengthen the ribosome assembly and prevent apoptosis triggered by cytochrome c (Cyt c). Our discovery opens a potential and novel paradigm of tRNA demethylase, which regulates biological functions via generation of tDRs.

Figure 1.

ALKBH3 localizes in cytoplasm and links to tRNA in vivo. (A) Immunofluorescence staining of endogenous ALKBH3 (red) was performed using anti-ALKBH3 antibody. Nuclei were counter-stained by DAPI (blue); (B) the relative enrichment folds of identified RNA types in ALKBH3-CLIP samples compared to that of the total input samples (CLIP/input of rRNA was used for normalization of other RNAs); (C) ALKBH3-bound RNAs in HeLa cells. Bakers yeast tRNA (Sigma 10109495001) and purified human tRNA were used as size marker. IgG was used as the negative control; (D) pie chart showing the identified top 20 specific tRNAs in ALKBH3-CLIP samples and input samples (two replicates for each group). See also Supplementary Figure S1.

Figure 2.

ALKBH3 catalyzes demethylation of m¹A and m³C in tRNA. (A) Total tRNAs were purified from HeLa cells and incubated with ALKBH3 protein or ALKBH3 protein + EDTA for 1 h (EDTA chelates cofactor iron and inactivates ALKBH3); (B) Michaelis–Menten plot of the steady-state kinetics of ALKBH3-catalyzed demethylation in stem-loop structured RNA probes that mimic the TΨC loops of tRNA^His(GUG), linear RNA probe and tRNA purified from HeLa cells at pH 7.5 at 37°C; (C, D) HeLa cells transfected with pcDNA (Vector) or pcDNA/ALKBH3 for 24 h, or siRNA negative control (si-NC) and si-ALKBH3–1∼3 for 48 h. Total tRNAs were then purified and digested for HPLC-MS/MS measurements of m¹A (C) and m³C (D); (E, F) The relative levels of m¹A (E) and m³C (F) of total tRNAs from lung, testis and epididymis of wide type or Alkbh3^−/−mice. Data were presented as means ± SD from three independent experiments. *P < 0.05 compared with control; **P < 0.01 compared with control. See also Supplementary Figure S2.

Figure 3.

Characterization and catalytic properties of ALKBH3 on specific tRNA. (A) In vitro biochemical assays of ALKBH3 wild type (WT), mutant R122A or mutant L177A in total tRNA of HeLa cells at 37°C for 1 h (EDTA chelates cofactor iron and inactivates ALKBH3); (B) the m¹A levels of tRNA in cytoplasm and nucleus isolated from wild type (WT) and Alkbh3^−/− HeLa cells, respectively; (C) the m¹A levels of tRNA isolated from the translation initiation non-polysome fractions (<80S, containing 40S ribosome, 60S ribosome and 80S monosome) and translation activing fraction (>80S, containing polysomes) from wild type (WT) and Alkbh3^−/− HeLa cells, respectively; (D) levels of m¹A in specific tRNA from wild type (WT) and Alkbh3^−/− HeLa cells, pulled down by corresponding cDNA probes; (E) levels of m³C in specific tRNA from wild type (WT) and Alkbh3^−/− HeLa cells, pulled down by corresponding cDNA probes; Data were presented as means ± SD from three independent experiments. *P < 0.05 compared with control. **P < 0.01 compared with control. See also Supplementary Figure S3.

Figure 4.

The promotion effects of ALKBH3 on cancer progression. (A) The relative cell proliferation of wild type (WT) vs Alkbh3^−/− HeLa cells were measured by CCK-8 kit; (B) cells were seeded in soft agar in a 96-well plate, grown for a week and soft agar colony formation was measured; (C) in vitro invasion assay of wild type (WT) versus Alkbh3^−/− HeLa cells for 24 h; (D) tumor growth curves of wild type (WT) versus Alkbh3^−/− HeLa cells in xenograft models at the indicated time interval; (E) IHC (Ki67)-stained paraffin-embedded sections obtained from xenografts. Red bar = 50 μm. (F) The wild type (WT) or Alkbh3^−/− HeLa cells were replaced with methionine-free medium supplemented with methionine analog HPG and incubated for 1 h. HPG incorporation was measured by confocal microscope, quantitatively analyzed the intensity of fluorescence, and normalized to nucleus (NuclearMask); (G) The polysome profiling of wild type (WT) or Alkbh3^−/− HeLa cells were analyzed; (H) Representative scheme of the reporter assay: the RNA reporter vector encodes firefly luciferase (F-luc) as the primary reporter and Renilla luciferase (R-luc) on the same plasmid as the internal transfection control. 6× CAC (His), 6× GGC (Gly) or 6× CAG (Gln)-coding sequences (recognized by tRNA^HisGTG, tRNA^GlyGCC and tRNA^GlnCTG, respectively) were inserted after the PLK promoter region. (I) Positive and control reporter were transfected into wild type (WT) or Alkbh3^−/− HeLa cells for 24 h. The control reporter without any insertion was used to normalize the translation differences between the two cells lines. Data were presented as means ± SD from three independent experiments. The cell proliferation was replicated six times for each group. *P < 0.05, **P < 0.01 compared with control. See also Supplementary Figure S4

Figure 5.

ALKBH3 catalyzes generation of tDRs via an ANG-dependent manner. (A) All smRNAs from WT or Alkbh3^−/-mice epididymis were purified and subjected to Illumina ultra-high–throughput sequencing (n = 2). The smRNAs were mapped to miRNAs, tRNAs, rRNAs or mRNA transcripts and normalized to the total reads; (B) pie chart showing the identified top 12 individual tDRs in WT or Alkbh3^−/-mice epididymis; (C) the 5′tDR-GlyGCC of lung, testis and epididymis from WT or Alkbh3^−/-mice was detected by northern blot analysis using biotin labeled probe (left) and quantitatively analyzed (right); (D) major cleavage sites and their corresponding percentages of cleaved tRNA^GlyGCC in wild type mice epididymis; (E) after transfected with si-ANG or negative control (si-NC) for 24 h, the 5′tDR-GlyGCC from stably overexpressing ALKBH3 and control HeLa cells were detected by northern blot analysis using biotin labeled probe (left) and quantitatively analyzed (right); (F) the purified tRNA (500 ng) of WT or Alkbh3^−/-mice epididymis was further treated with 100 ng ANG for 2 h. The production of 5′tDR-GlyGCC was detected by northern blot analysis using biotin labeled probe (left) and quantitatively analyzed (right). Data were presented as means ± SD from three independent experiments. **P < 0.01 compared with control. NS, no significant. See also Supplementary Figure S5.

Figure 6.

tDRs are involved in ALKBH3-induced cancer progression. (A) Wild type (wt) or Alkbh3^−/− HeLa cells were transfected with scrambled control RNA or synthesized 5′tDR-GlyGCC (31 nt) with the final concentration of 50 nM for the indicated times, and the cell proliferation was detected by CCK-8 kit; (B) HeLa cells transfected with scrambled control RNA or synthesized 5′tDR-GlyGCC (31 nt) for 24 h were replaced with methionine-free medium supplemented with methionine analog HPG and incubated for 1 h. HPG incorporation was measured by confocal microscope, quantitatively analyzed the intensity of fluorescence, and normalized to nucleus (NuclearMask); (C) the ribosome fractions of HeLa cells were grouped to non-ribosomal mRNPs, 40S, 60S, 80S and polysome. The expression of 5′tDR-GlyGCC in 40S, 60S and 80S were measured by TaqMan qRT-PCR; (D) compared to the scrambled control RNA group, 18 extra proteins were precipitated by both 5′tDR-GlyGCC and 5′tDR-GlnCTG in HeLa cells; (E) after crosslinking, the RPS21-binding RNAs were pulled down by the specific antibody. The 5′tDRs in input and RPS-binding RNAs were measured by TaqMan qRT-PCR and normalized to the total RNA amount; (F) both cap-dependent translation (R-luc) and IRES-dependent translation (F-luc) were normalized with LacZ activity as transfection control. The IRES-dependent translation (F-luc) were compared between wild type (WT) and Alkbh3^−/− cells for all the three groups; (G) both 5′tDR-GlyGCC and 5′tDR-GlnCTG had statistically significant effects on the HCV IRES reporter in HeLa cells; (H) HeLa cells transfected with control RNA or 5′tDR-GlyGCC for 24 h were further treated with NaAsO₂ for 6 h, stained with annexin V and propidium iodide (PI), and analyzed by flow cytometry (left). The percentages of apoptotic cells (annexin V and PI double positive) were quantified (right); (I) the caspase 3 and cleaved caspase 3 in cells treated as (I) were measured by western blot analysis. Data were presented as means ± SD from three independent experiments. The reporter assays were replicated for six times for each group. *P < 0.05, **P < 0.01 compared with control. See also Supplementary Figure S6.

Figure 7.

Proposed model of tDRs-mediated cancer progression triggered by ALKBH3. ALKBH3 catalyzed m¹A and m³C demethylation, which increases sensitivity of tRNA to ANG cleavage, leading to the formation of tDRs. ALKBH3-generated tDRs triggers the ribosome assembly and interacts with Cyt c to prevent cell apoptosis.