Biotin long arm maleimide

MOs were synthesized from Gene Tools LLC, and all of them were stored at −80°C in a concentration of 20 μg/μl (see table S1). Before microinjection, MOs were heated at 65°C for at least 10 min (95°C for 1 min for 3′tR-MOs), snapped on ice, and then diluted to the required concentration. Short RNA oligos/mimetics were synthesized from GenScript Biotech, and all of them were stored at −80°C in a concentration of 0.6 mM (table S2). Digoxigenin-labeled LNA-DNA chimeric oligos were synthesized from GenScript Biotech and stored at −20°C. DNA oligos, including primers, were synthesized by GENEWIZ (table S3). Biotinylated RNA oligos were synthesized using a 5’EndTag kit (Vector Laboratories, MB-9001), which was performed according to the manual. Biotin (Long Arm) maleimide (Vector Laboratories, SP-1501-12) was used for labeling a long-armed biotin group to the 5′ end of RNA oligos (GFP-r1 or 5′tRFl^Glu/CTC-mimetic), which was then purified by phenol:chloroform extraction. mRNAs for GFP and NLS-hRNASEH1-GFP were in vitro synthesized using T7 polymerase. The coding sequence of hRNASEH1 was amplified from HeLa cell RNAs using a pair of specific primers. MO or RNA was injected into the yolk of zebrafish embryos at the 1c stage. The injection doses are given in the figures or figure legends.

Purified RNA was dephosphorylated using calf intestine phosphatase (Roche). For 5′ end biotinylation, 600 pmol RNA were incubated with 0.2 mM γ-S-ATP (Biomol) and T4 polynucleotide kinase (New England Biolabs) for 30 min at 37°C. Biotin-long-arm maleimide (Vector Laboratories) was added and incubated for 30 min at 65°C. Unincorporated label was depleted by lithium chloride precipitation. Biotinylated RNA (200 pmol) was conjugated to Dynabeads M-280 (Invitrogen) in incubation buffer (10 mM Tris–HCl pH 7.4, 150 mM potassium chloride, 0.5 mM DTT, 0.05% NP40, 100 U/ml RNasin) for 2 h at 4°C with continuous rotation. Whole cell protein lysate (1 mg) of HEK293 cells together with 200 μg yeast tRNA (Sigma-Aldrich) and 5 mg Heparin (Sigma-Aldrich) was added to the beads and incubated 1 h at 4°C followed by 15 min at room temperature with continuous rotation. Beads were washed five times with incubation buffer, resuspended in 30 μl protein loading dye and boiled at 95°C for 10 min. Eluted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad). The primary antibody was anti-AUF1 (1:10 000; rabbit; Millipore; 07-260) and the secondary antibody was Horseradish-conjugated anti-rabbit (1:7000; goat; Jackson ImmunoResearch). Blots were developed using ECL Select (Life Technologies). Imaging was performed on a ChemiDoc Imaging system (Bio-Rad).

The template DNAs for in vitro transcription of Int2-1, Int2-2, Int2-3, Int2-1-2-1, Int2-1-2-2, Int2-1-2-2a, Int2-1-2-2b and Int2-1-2-2pSL RNA were amplified with KOD-Plus-Neo DNA polymerase using the Ex1-(Int2)-Ex4-GFP minigene as a template for Int2-1, Int2-2, Int2-3, Int2-1-2-1, Int2-1-2-2, Int2-1-2-2a and Int2-1-2-2b and Int2pSL for Int2-1-2-2pSL, respectively. Supplementary Table S1 lists the primer sets. Amplified DNA was separated by 1% agarose gel electrophoresis and purified from gel pieces using the QIAquick Gel Extraction kit. In vitro transcription was performed with 0.2 pmol of the template DNA using CUGA® 7 in vitro transcription kit (NIPPON GENE). Transcripts were collected with the RNeasy Mini kit (Qiagen) (Int2-1, Int2-2, Int2-3) or isopropanol precipitation (Int2-1-2-1, Int2-1-2-2, In2-1-2-2a, Int2-1-2-2b, Int2-1-2-2pSL) and purified using reverse-phase liquid chromatography. Biotin (BT) was added to the 5′ end of RNA using the 5′ EndTag Nucleic Acid End Labeling System and Biotin (Long Arm) Maleimide (Vector Labs). BT-RNA was collected by ethanol precipitation.