Pierce chemiluminescent nucleic acid detection module kit

Transcripts were generated employing the HiScribe™ T7 High Yield RNA Synthesis Kit (NEB, Frankfurt, Germany). Transcripts were biotinylated employing the Pierce™ RNA 3′ End Biotinylation Kit (NEB, Frankfurt, Germany) according to the manufacturer’s protocol. Briefly, 50 pmol of transcript was labeled by ligation with a single biotinylated nucleotide at the 3′-terminus and subsequently purified. Labeling efficiency of biotinylated RNA was determined by dot blotting whilst following the description of the Pierce™ Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Scientific, Vienna, Austria).
Proteins binding to the respective transcripts were isolated employing streptavidin magnetic beads (Thermo Scientific, Vienna, Austria) according to the manufacturer’s protocol. Briefly, beads were washed and supplemented with RNA Capture buffer. Then, 50 pmol biotin-labeled RNA was added to the beads, followed by an incubation for 30 min at RT with agitation. Protein-RNA binding buffer (Tris pH 7.5 20 mM, NaCl 50 mM, MgCl₂ 2 mM, Tween 0.1%(v/v)), 30% glycerol, and 20 μg of nuclear lysate were added to the beads and incubated for 60 min at 4 °C with agitation. RNA-binding protein complexes were collected and washed with wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole) and eluted in 50 mM ammonium acetate. Proteins were separated by SDS-PAGE and subsequently silver stained.

To detect RNAs being part of the identified protein complexes, northern hybridization assays were performed as previously described (Yoo et al., 2004). The BN‐PAGE gels were transferred (Fastblot 43B semi dry Blot; Biometra, Göttingen, Germany) onto a H‐bond nylon membrane (Amersham Hybond‐N+, GE Healthcare Life Sciences, Uppsala, Sweden), UV‐crosslinked (Stratalinker 2400, Agilent, Santa Clara, CA, USA) and prehybridized with ULTRAhyb^© Ultrasensitive Hybridization Buffer (Ambion^®; Life Technologies) for 1 h at 68°C. Then, 3′ biotinylated oligonucleotide probes (Supporting Information Table S1) were added and the membrane was incubated again at 68°C cooling down to 37°C for 12 h, before washing with 2× sodium saline citrate buffer (300 mM NaCl, 30 mM Na₃citrate pH 7.0) at room temperature for 2 min. RNAs were detected using Pierce Chemiluminescent Nucleic Acid Detection Module Kit (ThermoFisher Scientific).

The hybridization probe for the southern blot analysis of the TDR transformants was designed to consist of a single fragment containing FADO, TprC terminator, tef1 promoter and FAR sequence, originating from the previously constructed expression vector (Fig. 3a). The DNA probe was biotin labeled by using Pierce North2South Biotin Random Prime Kit (Thermo Fisher Scientific, Rockford, IL, USA) according the manufacturer’s protocol. Genomic DNA of the transformants was extracted as described above and digested with restriction enzymes BamHI, PstI, and NdeI (all purchased from Thermo Scientific, Rockford, IL, USA). Southern blotting was carried out using Whatman Turboblotter transfer system (GE Healthcare Life Sciences, Pittsburg, PA, USA) and the nucleic acid detection was carried out by using Pierce Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific, Rockford, IL, USA), both according to the manufacturer’s protocol.