Pcmv6 an mrfp

MAPKBP1 and WDR62 plasmids pcDNA3.1D_JNKBP1/V5-6xHis-TOPO and pCAN3_WDR62 were previously published.^{1 (link)} For WDR62, N-terminal myc-was replaced by HA- and for MAPKBP1 C-terminal V5- by 2xDDK- or HA-tag sequences using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA). For G1442Vfs*12, intronic bases were introduced, whereas E1112* was constructed by introduction of a stop codon at amino acid position 1112 (Supplementary Data S2). For N-terminal GFP-tags the GFP-sequence of TagGFP2-hPC2 (P. Harris and V. E. Torres, Mayo Clinic, Rochester, MN) was amplified with primers containing HindIII restriction sites. Polymerase chain reaction products and plasmids were ligated with T4 DNA Ligase after digestion (Thermo Fisher Scientific, Waltham, MA). To generate N-terminally RFP-tagged MAPKBP1, pCMV6-AN-mRFP (OriGene Technologies, Rockville, MD) and pcDNA3.1D_JNKBP1/2xDDK-His-TOPO were digested with FastDigest MssI and FastDigest HindIII (FD1344; Thermo Fisher Scientific). Site-directed mutagenesis polymerase chain reaction was performed to restore the correct reading frame. Sequences were verified by Sanger sequencing. Plasmids encoding p-EGFP-G3BP+STOP, mRFP-Dcp1a, and mRFP-TIA1 were kindly provided by N. Kedersha (Brigham and Woman’s Hospital, Boston, MA).^{28 (link)}

pCMV6-AN-Myc-DDK (PS100016; RRID:SCR_021264) and pCMV6-AN-mRFP (PS100049; RRID: SCR_021265) were purchased from Origene. Inducible GFP-RNAseH1 plasmid was generated via restriction cloning an RNH1-GFP cassette from pEGFP-RNASEH1, a gift from Andrew Jackson & Martin Reijns (RRID:Addgene_108699) using BglII + NotI and cloning into the BamH1 NotI sites of pENTR1A (RRID:SCR_021263). The RNH1-GFP cassette was then transferred to the pCLX-pTF-R1-DEST-R2-EBR65 lentiviral plasmid, a gift from Patrick Salmon (RRID:Addgene_45952) via an LR recombination reaction, creating a single vector that expresses the Tet-repressor and blasticidin resistance gene under the control of a constitutive promoter and containing the RNHA1-GFP cassette under the control of a Tet-repressor regulated promoter. SF3B1-K700E and RNAseH1 D210N mutations where created by site-directed-mutagenesis.