Cell lines and cell culture

Human immortalized uroepithelial cells SV-HUC-1, 293T cells and human bladder cancer cells T24 were purchased from the American Type Culture Collection (Manassas, VA, USA) and the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). In this study, cells were cultured using complete media containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin–streptomycin. SV-HUC-1 cells were cultured in Ham's F-12 K (Kaighn's) medium (Gibco, USA), T24 cells were cultured in RPMI-1640 medium (Gibco, USA), and 293T cells were cultured in DMEM (Gibco, USA) medium. The cell culture parameters were set to 5% CO2 and 37 °C. All cell lines were free of mycoplasma infection.

Stressors and treatment

CdCl2 (Sigma, USA) and NaAsO2 (Sigma, USA) were dissolved in ddH2O to a concentration of 10 μmol/ml and stored long-term at −80 °C. Before treating cells, fresh culture medium was used to dilute CdCl2 and NaAsO2 to the required concentrations, pre-warmed to 37 °C, and then added to the cells for culture for an appropriate amount of time.

Immunofluorescence and statistical analysis of SGs

Cells were grown in 8-well chamber slides (Millipore, USA). Add 300 M CdCl2/NaAsO2 for 1 h stimulation. Subsequently, the cells were washed three times with PBST buffer (0.1% Tween) at 37 ℃ for 5 min each time. The cells were then fixed with 4% paraformaldehyde (Sihe Biotech, China) at room temperature for 20 min, followed by three washes with PBST buffer. The cells were permeabilized with 0.5% Triton X-100 for 5 min, and then washed three times with PBST buffer. Next, blocked with 1% bovine serum albumin (BSA) for 1 h. Cells were further incubated with primary antibodies in 1% BSA overnight at 4 ℃, followed by three washes with PBST buffer. Then, cells were incubated with secondary antibody at room temperature for 1 h, avoiding exposure to light. After three washes with PBST buffer, the cells were incubated with DAPI for 10 min and then washed three times with PBST buffer, avoiding exposure to light. Images were captured by ZEISS/LSM880 confocal microscope (Zeiss, Germany).

To perform statistical analysis on the photographed images, the positive cell rate for the production of SGs can be calculated using the following formula: \(Positive\;Cell\;Rate=\frac\times100\%\). The antibodies used are shown in Table S1(Supplementary file 1).

Plasmid construction and mutagenesis assay

The cell lines for overexpression/knockdown of METTL1 have been previously constructed in the laboratory (Ying et al. 2021). The plasmid for overexpressing G3BP1-EGFP was obtained by cloning the target gene G3BP1-EGFP cDNA from the phage UbiC G3BP1-EGFP-EGFP plasmid (Addgene 119950) and then ligating it with LentiORF PLEX-MCS. The primers used are listed in Table S2(Supplementary file 1). The G3BP1 mutant fragment was synthesized by Azenta Life Sciences, and the synthesis report is provided in Supplementary File 7. The cDNA of G3BP1 wild-type and mutant were ligated into 2AB-Flag vector. For detailed experimental methods, please refer to Supplementary File 1.

Western blot

The cells were lysed with RIPA buffer (Beyotime, China) containing 1 × PMSF. After determining the protein concentration using the BCA protein assay kit (Beyotime, China), the protein sample was mixed with 5 × SDS-PAGE loading buffer (Beyotime, China) and boiled at 100℃ for 10 min. The sample was separated by 10% SDS-PAGE gels and transferred onto a polyvinylidene difluoride membranes (Biorad, USA). The membrane was blocked with 5% BSA at room temperature for 1 h, and then incubated with the primary antibody overnight at 4 ℃. After washing the membrane three times with TBST (0.1% Tween) for 10 min each time, the membrane was incubated with the secondary antibody at room temperature for 1 h and then washed three times with TBST. The immune response was visualized by using ECL chemiluminescence reagent kit (Thermo Fisher, USA), and the image was captured using iBright™ FL1500 Imaging System (Thermo Fisher, USA). The antibodies used are shown in Table S1.

RNA isolation and qRT-PCR

After extracting total cellular RNA using Trizol reagent (Invitrogen, USA), the RNA was reverse transcribed into cDNA using PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Japan). According to the manufacturer’s instructions, a mixture containing mix, cDNA, and primers were prepared. The cDNA in the mixture was quantified using a Step-One Fast Real-time PCR System(Applied Biosystems, USA), and the data were calculated using the 2 -△△CT method. The primers used are listed in Table S2.

Sucrose gradient centrifugation and polysome fractionation

6 × 107 cells were added 100 μg/mL CHX (Sigma, USA) and cultured at 37 °C for 15–20 min. Then the cells were washed twice with pre-cooled PBS and collected in a centrifuge tube. The tube was put on ice and cells were lysed with polysome cell extraction buffer (50 mM MOPS, 150 mM NaCl, 15 mM MgCl2, 0.5% Triton X-100, 100 μg/ml Cycloheximide, 200 U RNaseOUT, 1 mg/ml Heparin, 2 mM PMSF, and 1 μM Benzamine) for 10 min. After centrifugation (12,000 × rpm, 15 min, 4 °C), supernatant was collected and added to a 10%−50% sucrose gradient prepared with sucrose and polysome buffer (0.05 M MOPS, 0.015 M MgCl2, 0.15 M NaCl, 0.1 mg/mL CHX). Monosomes and polysomes were separated by using the SW41 rotor (Beckman Coulter, USA) to centrifuge at 36,000 rpm for 2.5 h at 4 °C. mRNA was purified and reversed transcription, then an equal volume of cDNA for qRT-PCR analysis.

Extraction for SG core RNA

Extraction for SG core RNA was performed as previously described (Khong et al. 2018). In brief, 6 × 107 cells were cultured in fresh pre-warmed culture medium containing 300 μM CdCl2 at 37 °C for 1 h. After that, the cells were washed three times with fresh pre-warmed culture medium at 37 °C and collected in a 50 mL Eppendorf tube. The tube was centrifuged at 1500 g for 3 min and the supernatant was discarded. The cell pellet was then transferred to liquid nitrogen for rapid freezing overnight. The next day, the cell pellet was thawed on ice for 30 min and SG lysis buffer was added to fully lyse the cell pellet. The supernatant (SG core-enriched fraction) was collected through multiple centrifugations. The SG-enriched fraction was pre-treated with Pierce™ Protein A/G Magnetic Beads (Thermo Fisher, USA) balanced with SG lysis buffer containing DEPC. Subsequently, GFP antibody was added and incubated overnight at 4 °C with rotation. The mixture was then centrifuged at 18,000 × g for 20 min at 4 °C, the supernatant was removed, and magnetic beads was added. The mixture was rotated at 4 °C for 180 min. The beads were washed with Wash buffer 1, Wash buffer 2 and Wash buffer 3, followed by resuspension in 250 μL of 1 × Proteinase K buffer and incubated at 37 °C for 15 min. Finally, 750 μL of Trizol was added to purify the SG core RNA. The components of the buffers used are provided in Supplementary Material 1.

tRF & tiRNA sequencing

The tRF & tiRNA sequencing was conducted by Kangcheng Biotech (Guangzhou, China). Purified RNA was extracted and assessed for quality using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher, USA). To eliminate RNA modifications that could interfere with small RNA-seq library construction, total RNA samples were subjected to the following pretreatments: deacylation of 3'-aminoacyl (charged) groups to generate 3'-OH for 3'-adapter ligation; removal of 3'-cP (2',3'-cyclic phosphate) to produce 3'-OH for 3'-adapter ligation; phosphorylation of 5'-OH (hydroxyl group) to create 5'-P for 5'-adapter ligation; and demethylation of m1A and m3C to facilitate efficient reverse transcription. Library preparation involved ligating 3' and 5' small RNA adapters to both ends of the total RNA from each sample, followed by synthesis and amplification of cDNA using Illumina-specific RT reverse transcription primers and amplification primers. Subsequently, PCR amplicons of ~ 134-160 bp were extracted and purified from PAGE gel. Finally, the completed libraries were quantified using the Agilent 2100 Bioanalyzer. The libraries were denatured and diluted to a loading concentration of 1.8 pM in a final volume of 1.3 mL. The diluted libraries were then loaded onto the reagent cartridge according to the Illumina NextSeq 500/550 V2 Kit (FC-404–2005, Illumina, USA) and run on the Illumina NextSeq 500 system (Illumina, USA). Data were collected and analyzed.

Image analysis and base calling are performed using Solexa pipeline v1.8 (Off-Line Base Caller software, v1.8). Sequencing quality are examined by FastQC (Andrews 2010) and trimmed reads (pass Illumina quality filter, trimmed 5, 3 -adaptor bases by cutadapt (Martin 2011)) are aligned allowing for 1 mismatch only to the mature tRNA sequences, then reads that do not map are aligned allowing for 1 mismatch only to precursor tRNA sequences with bowtie software (Langmead et al. 2009). The remaining reads are aligned allowing for 1 mismatch only to miRNA reference sequences with miRDeep2 (Friedländer et al. 2012). The expression profiling of tRF & tiRNA and miRNA can be calculated based on counts of reads mapped. The differentially expressed tRFs & tiRNAs are screened based on the count value with R package edgeR (Robinson et al. 2010).

tiRNAs were detected by qRT-PCR

Total RNA from cells was extracted using Trizol reagent. According to the rtStar™ tRF & tiRNA Preatreatment Kit (Arraystar, USA) manufacturer’s instructions, the total RNA was pre-treated, including 3' terminal deacetylation, demethylation, 3'-cP removal, 5'-P addition, and 5' adaptor ligation. Following the PrimeScript™ RT reagent Kit (Perfect Real Time) (Takara, Japan) manufacturer’s instructions, the pre-treated RNA was reverse transcribed into cDNA for subsequent qRT-PCR detection. The primers used are listed in Supplemental Table S2.

Northern blot

Northern Blot was performed as previously described (Edmonds et al. 1991). Briefly, 6 μg total RNA was separated by electrophoresis (100 V, 2 h) on a 15% urea PAGE, followed by immediate transfer onto a nylon membrane (Thermo Fisher, USA) (30 V, 2 h). Those processes were performed on ice. After drying the nylon membrane, it was UV cross-linked for 3 min on each side with an energy of 2400. The nylon membrane was then incubated with Buffer A at 37 °C for 30 min, followed by overnight incubation with the 3’-DIG–labeled probes (Sangon Biotech, China) at 37 °C. The membrane was first washed with Low Stringent Buffer, High Stringent Buffer and Washing Buffer at 37 °C. After the washes, the membrane was blocked with Blocking Buffer at room temperature for 3 h, and then incubated with DIG antibody (Sigma, USA) at room temperature for 30 min. The membrane was then washed with DIG Washing Buffer. Finally, the membrane was incubated with the luminescent substrate CSPD (Roche, Switzerland) at 37 °C in the dark for 20 min, and the luminescent signal was visualized using an imaging system. The probes and the buffers used are listed in Supplementary file 1.

tiRNA interference and overexpression

Transfections of tiRNA inhibitor were performed using Lipofectamine® RNAiMAX Reagent (Thermo Fisher, USA) manufacturer’s protocols. Transfections of tiRNA mimics and endogenous tiRNA were performed using Lipofectamine® 3000 Transfection kit (Thermo Fisher, USA) manufacturer’s protocols. The inhibitors and mimics were synthesized by Sangon, and the sequences are provided in Supplementary file 1.

Extraction for endogenous tiRNA

6 × 107 cells were washed twice with 1 × HBSS and collected. The cells were centrifuged at 4 ℃ for 3 min at 1000 g. 250 μL of ANG digestion buffer (5 mM MgCl2, 0.5% NP-40, 1 × PBS pH7.4) was added to the cells, followed by centrifugation at 4 ℃ for 30 s at 1500 g to collect the supernatant. 100 nM angiogenin (ANG) (Novoprotein, China) was added to the supernatant and incubated at room temperature for 30 min. RNA was extracted using Trizol. Then the RNA was separated by electrophoresis (100 V, 2 h) on a 7.5% urea PAGE. The gel was transferred to a 3 × SYBR GelRed solution (Solarbio, China) and incubated at room temperature for 1–1.5 h. The gel containing tiRNA fragments was cut under a UV lamp. 1 × Elution buffer (20 mM Tris–HCl pH7.4, 250 nM CH3COONa, 1 mM EDTA, 0.25% SDS) was added to the gel and incubated at 65 ℃ for 15 min, followed by freezing at −80 ℃ for 15 min, and another incubation at 65 ℃ for 15 min. The supernatant was then collected and RNA was extracted using Trizol. 500 pmol biotinylated oligo probe (Ribiotech,China) and 950 μL of TBS buffer (0.05% NP-40, 200 U RNase Inhibitor) were added to the RNA and incubated at room temperature for 1 h. Streptavidin magnetic beads (Thermo Fisher, USA) washed three times with TBS buffer, were added and incubated at room temperature for 1 h. The magnetic beads were washed three times with TBS buffer for 5 min each time, and then Trizol was added to purify the RNA-probe complex. After digestion with DnaseI, the Trizol was added to extract RNA. The RNA was incubated at 4 ℃ for 2 h with m7G antibody (RNA: m7G antibody = 1:10) and 500 μL of 1 × IPP buffer (2 mM Tris–HCl pH7.4, 30 mM NaCl, 0.02% NP-40), followed by overnight incubation at 4 ℃ with Pierce™ Protein A/G Magnetic Beads (Thermo Fisher, USA) washed with 1 × IPP buffer. After washing the beads three times with 1 × IPP buffer, Trizol was added to extract the RNA, resulting in endogenous m7G 3' tiRNA.

Statistical analysis

The data of 3 independent experiments were statistically analyzed (all with mean ± standard deviation). The software Graphpadprism8 (GraphPad, CA, USA) and SPSS 16.0 (IBM, Armonk, NY, USA) was used to analyze and plot the experimental data. The statistical methods were adjusted according to the type of experiment. Inspection level of α = 0.05, P < 0.05, considered statistically significant, this paper identifies the NC: P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, ****P < 0.0001.