Kod plus mutagenesis kit

[Methyl-³H] SAM (78.0 Ci/mmol) was purchased from PerkinElmer Inc. (Waltham, MA, USA); [³H] DMAPP (20.0 Ci/mmol) was obtained from BIOTREND Chemicals (USA); SAM was purchased from NEW ENGLAND Biolabs Inc..; Dimethylallyl diphosphate (DMAPP), dithiothreitol (DTT), NTPs, 5′-GMP, pyrophosphate, Tris-base, β-mercaptoethanol (β-Me), MgCl₂, NaCl and KCl were purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA); Isopropyl β-D-thiogalactoside (IPTG) was obtained from AMRESCO (OH, USA); Nitrocellulose membranes (0.22 μm) were purchased from Merck Millipore (Darmstadt, Germany); Primers for PCR were synthesized by BioSune (Shanghai, China); Nickel-nitrilotriacetic (Ni-NTA) Superflow was purchased from Qiagen Inc.. (Germany); KOD-plus mutagenesis kit, Pyrobest DNA polymerase and the dNTP mixture were obtained from Takara (Japan); T4 polynucleotide kinase, T4 DNA ligase, Ribonuclease inhibitor and all restriction endonucleases were obtained from Fermentas/Thermo Scientific (MA, USA); The E. coli JW3581-1 and JW4129-2, strains in which the gene of TrmL and MiaA were deleted respectively, were purchased from the E. coli genetic stock center (Yale University, New Haven, CT, USA); PrepHT 300SB-C18 was purchased from Agilent Technologies (CA, USA).

To construct the expression plasmids of Flag-ZFC3H1 full length (FL) or fragments, the corresponding sequences were cloned into Flag-Phage using ClonExpress^® Ultra One Step Cloning Kit (Vazyme Biotech Co., Ltd). The deletion or point mutation ZFC3H1 expression plasmids were constructed by mutagenesis using the KOD-Plus-Mutagenesis Kit (Takara). ZFC3H1-FL-ZsGreen and ZFC3H1-NP-ZsGreen were constructed by deletion of the sequence between ZFC3H1 and ZsGreen in Flag-ZFC3H1-FL and Flag-ZFC3H1-NP, respectively. Plasmids encoding Flag-eIF4A3 and Flag-DDX3 were described previously (20 (link),25 (link)). To generate construct for gene editing, sgRNA targeting ZFC3H1 was synthesized, annealed and ligated to the pX330-mCherry plasmid. The sgRNA sequence is shown in Supplementary Table S1. The Smad and β-globin constructs were described previously (28 (link)).
The CBP80, MTR4, UAP56 and ARS2 antibodies were described previously (20 (link)). Antibodies against PABPN1 (Abcam), ZFC3H1 (Novus), Flag (Sigma-Aldrich), GAPDH (Proteintech), Tubulin (Proteintech), SC35 (Sigma-Aldrich), digoxin (Roche) and SON (Thermo Fisher) were purchased. Alexa Fluor 546, Alexa Fluor 488 or Alexa Fluor 647 conjugated secondary antibodies were purchased from Life Technologies.

SAM, SAH, 5′-GMP, Tris-base, β-mercaptoethanol (β-Me), KCl and the reagents used to optimize crystallization conditions were purchased from Sigma–Aldrich (St Louis, MO, USA). MgCl₂, NaCl, adenosine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate were from Sangon Biotech (Shanghai, China). [Methyl-³H] SAM was obtained from PerkinElmer (Waltham, MA, USA); crystallization kits were from Hampton research (Aliso Viejo, CA, USA). Primers for polymerase chain reaction were synthesized by Invitrogen (Shanghai, China) and Biosune (Shanghai, China); the pET22b vector was from Merck–Millipore (Darmstadt, Germany). The KOD-plus mutagenesis kit, Pyrobest DNA polymerase and dNTP mixture were obtained from Takara (Shiga, Japan); T4 ligase and other restriction endonucleases were obtained from MBI Fermentas (Pittsburgh, PA, USA). Pyrophosphatase (PP_iase) was purchased from Roche Applied Science (Basel, Switzerland). Ni²⁺-NTA Superflow was obtained from Qiagen (Dusseldorf, Germany). The Superdex 200 column and 3 mm filter papers were from GE Healthcare (Fairfield, CT, USA). The analog of SAM, sinefungin, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).