Fastap phosphatase

COX2 mRNA 5′-UTR was obtained in in vitro transcription reaction by T7 RNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) with annealed corresponding oligonucleotides as the template. After the reaction, the mix was digested with DNAse I (Thermo Fisher Scientific, USA) and then loaded on 8% denaturing PAAG. After electrophoresis, band corresponded to COX2 mRNA 5′-UTR was excised, and RNA was passively eluted in 1 mL 20 mM Tris-HCl pH 7.5, 250 mM NaOAc, 1 mM EDTA, and 0.25% SDS overnight at room temperature. Subsequently, RNA was precipitated, dephosphorylated by FastAP phosphatase (Thermo Fisher Scientific, USA), and then labeled with γ-³²P-ATP with polynucleotide kinase (Thermo Fisher Scientific, USA).
Binding reactions were performed in 20 µL of 20 mM Tris-HCl pH 7.2, 50 mM NaCl, 5 mM MgCl₂, 5% glycerol, each reaction mix contained 50 fmol of labeled COX2 mRNA 5′-UTR, 0.5 pmol of unlabeled total yeast tRNA, and different concentrations of recombinant Pet111p and/or Aim23p. After binding for 20 min at room temperature, the reaction mixes were loaded on 8% PAAG at 1× TBE and separated at 100 V for 1 h. Afterwards, the gels were dried and subjected to autoradiographic analysis using Storm 865 scanner (GE Healthcare, USA).

The R418 RNA transcripts were 5′ dephosphorylated using the FastAP phosphatase (Thermo) and then 5′-end-³²P labeled with T4 polynucleotide kinase (NEB) in the presence of γ-³²P ATP and purified through Microspin G25 columns (GE healthcare). RNase assay was carried out at 30°C in 50 mM Tris pH 7.5, 150 mM KCl, 5 mM DTT with 250 nM of the R418 ³²P-labeled RNA transcripts and 1 μM R343+H₆ in the presence of 5 mM MgCl₂ or 0.5 mM MnCl₂. Where indicated, 1 μM of H6c-R341 was added to the reaction. Reaction products were separated on 8 M urea-10% polyacrylamide gel.

Detailed information regarding the plasmids generated in this study are provided in Supplementary file 5A. Plasmids generated in this study have been produced (i) by ligating annealed primers into BbsI digested plasmids (for cloning of sgRNAs) or (ii) by ligating digested PCR-amplified cDNAs into dephosphorylated plasmids. Phusion DNA polymerase, FastDigest restrictions enzymes and FastAP phosphatase were used (Thermo Fisher Scientific). For all plasmids, DNA sequencing (Mix2Seq, Eurofins Genomics) was used with the specified primers, to confirm that the desired sequence was inserted. All new plasmids have been deposited on Addgene. The plasmids ERmox-GFP (Addgene #68072, Costantini et al., 2015 (link)), Ub-G76V-YFP (Addgene #11949, Menéndez-Benito et al., 2005 (link)), mCherry-Mito-7 (Addgene #55102, Olenych et al., 2007 (link)), pEGFP-N1-ATG-FLAGC (Addgene #60360, Britton et al., 2014 (link)), pEGFP-C1-FLAGN (Addgene #46956, Britton et al., 2013 (link)), pICE-EGFP-FLAG-Ku70siR-WT (Addgene #46961, Britton et al., 2013 (link)), and pCAG-eSpCas9-2A-GFP (Addgene #79145) were provided by Addgene thanks to Addgene contributors.