Positively charged nylon membrane

The 20 μg total RNA or encapsidated pgRNA was resolved by electrophoresis on 1.2% formaldehyde agarose gel and transferred to a positively charged nylon membrane (Thermo Fisher Scientific). The membrane was fixed by UV cross-linking. The fixed membrane was prehybridized for 30 min and then incubated by ULTRAhyb hybridization buffer (Invitrogen) for 30 min and then hybridized with an internally radiolabeled double-stranded DNAprobe in ULTRAhyb hybridization buffer (Thermo Fisher Scientific) overnight. The probe was generated using [α-³²P] dCTP and DECAprimeII kit (Thermo Fisher Scientific). After, washing, the membrane was exposed with BIOMAX XAR film (Sigma-Aldrich). The probe template was amplified by PCR using forward (5′- ATGGCTGCTAGGCTGTGCTGCC-3′) and reverse (5′-ATAAGGGTCGATGTCCATGCCCCAAAG-3′) primer from the pHBV1.3-mer plasmid.

To assess the effect of RNA mutation on binding of RBP, electrophoresis mobility shift assays were performed using LightShift Chemiluminescent RNA EMSA kit (Catalog # 20158; Thermo Scientific, Rockford, USA). Two purified RBPs were used in the assay. Of which, PCBP3 was purchased from OriGene Technologies (Catalog # TP329176; Rockville, USA), and PTBP1 was kindly provided by Dr Yuanchao Xue (Institute of Biophysics, Chinese Academy of Sciences, Beijing, China). Then, 200 ng RBP was pre-incubated with 100 μg ml⁻¹ tRNA in 1× RNA EMSA binding buffer for 10 minutes at room temperature. After that, 80 fmol synthesized 3′-biotin-labeled wild-type or point-mutated RNA oligos (Supplementary Table S2) were respectively added to the mixture (15 μl final volume) and incubated for 20 minutes at room temperature. Then, 3.75 μl 5× loading buffer was added to the 15 μl RBP-RNA mixture and immediately loaded into the pre-run TBE polyacrylamide gel, and ran at 100 V for 45–60 min in cooled 0.5× TBE buffer. Samples were then transferred to positively charged nylon membrane (Thermo Scientific, Rockford, USA), and crosslinked with UV-light crosslinking instrument equipped with 254 nm bulbs. The subsequent blocking, washing and detection were performed according to the manufacturer's instructions.

tRNA fragmentation was examined by Northern blot adapting a previously described protocol (44 (link)). 10 μg short (<200 bp) RNA was denatured in formamide and run in 15% polyacrylamide-8 M urea gels at 120V for 2–3 h. RNA was transferred to positively charged nylon membrane (ThermoFisher) through electroblot transfer performed at 250 mA for 2 h, followed by 350 mA for 1 h at 4°C in 0.5× TBE. Membranes were crosslinked by short-wave UV light, pre-hybridized for 2 h in hybridization solution (5x SSC, 1x Denhardt's solution (Sigma Aldrich), 7% SDS, 20 mM Na₂PO₄) at 46°C, and then hybridized overnight at 46°C with 10 ng/ml [γ-³²P]ATP-labeled tRNA-specific DNA probes complementary to 5′ or 3′ halves of tRNAs. Probe sequences are provided in Supplementary Table S13. Membranes were washed in 2x SSC/0.1% SDS, 1x SSC/0.1% SDS and 0.1x SSC, air-dried and radioactive signal detected by autoradiography.