The Roquin ROQ domain used in this study was expressed from the pETTrx1a vector, obtained from Gunther Stier (EMBL); its cloning has been described before (45 (link)). For protein purification of the RNA recognition motifs of AUF1 (AUF1_RRM2 and AUF1_RRM1-2), coding sequences were generated by PCR amplification using pCMV-hnRNP D37 as a template (47 (link)) and introduced into XbaI and XhoI sites of an His6- and thioredoxin-tag encoding plasmid based on pETTrx1a, i.e. including a TEV (tobacco etch virus) site for proteolytic removal of all tags.
For RNA synthesis, CDE1, CDE2, CDE1GG, CDE2GG and CDE1-2 (tandem) sequences together with the T7 promoter were generated by hybridization of complementary oligonucleotides and introduced into the NcoI and HindIII sites of an HDV ribozyme encoding plasmid based on the pSP64 vector (Promega). RNAs were transcribed as HDV ribozyme fusions to obtain uniform 3′ ends.
For the luciferase reporter system, 3′-UTR variants were generated by hybridization of complementary oligonucleotides and introduced downstream of the firefly luciferase open reading frame into the multiple cloning site of pDLP (48 (link)) using NotI and HindIII restriction sites.
All protein and RNA sequences are summarized inSupplementary Tables S1 and S2 .
For RNA synthesis, CDE1, CDE2, CDE1GG, CDE2GG and CDE1-2 (tandem) sequences together with the T7 promoter were generated by hybridization of complementary oligonucleotides and introduced into the NcoI and HindIII sites of an HDV ribozyme encoding plasmid based on the pSP64 vector (Promega). RNAs were transcribed as HDV ribozyme fusions to obtain uniform 3′ ends.
For the luciferase reporter system, 3′-UTR variants were generated by hybridization of complementary oligonucleotides and introduced downstream of the firefly luciferase open reading frame into the multiple cloning site of pDLP (48 (link)) using NotI and HindIII restriction sites.
All protein and RNA sequences are summarized in
