KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Rabbit polyclonal anti-eIF4E | MBL Lifescience | Cat# RN001P, lot 004; RRID:AB_1570634 |
| Rabbit polyclonal anti-eIF4G1 | MBL Lifescience | Cat# RN002P, lot 005; RRID:AB_1570635 |
| Rabbit polyclonal anti-vinculin | Cell Signaling Technology | Cat# 4650, lot 5; RRID:AB_10559207 |
| Rabbit polyclonal anti-DDX6 | Abcam | Cat# ab151984, lot GR174405-10; RRID:AB_3073919 |
| Rabbit polyclonal anti-CNOT3 | Bethyl Laboratories | Cat# A302-156A; RRID:AB_1720348 |
| Mouse monoclonal anti-FLAG | Millipore Sigma | Cat# F1804, lot SLCK5688; RRID:AB_262044 |
| Mouse monoclonal anti-FLAG (for Flag fabs, imaging) | FUJIFILM Wako Shibayagi | Cat# 012-22384, clone 1E6; RRID:AB_10659717 |
| Mouse monoclonal anti-GFP | Roche | Cat# 11814460001, lot 42903200; RRID:AB_390913 |
| Rabbit polyclonal anti-Renilla luciferase | Abcam | Cat# ab187338, lot GR3382572-1; RRID: AB_3073920 |
| Mouse monoclonal anti-hGAPDH | Proteintech | Cat# 60004-1-Ig, lot 10020246; RRID:AB_2107436 |
| Mouse monoclonal anti-Ubiquitin | Thermo Fisher Scientific | Cat# 14-6078-82, lot 4342660; RRID:AB_837154 |
| Rabbit polyclonal anti-Firefly Luciferase | GeneTex | Cat# GTX125849, lot 42837; RRID:AB_11173184 |
| Mouse monoclonal anti-HA tag | Thermo Fisher Scientific | Cat# 26183, lot WA317137; RRID:AB_10978021 |
| Mouse monoclonal anti-Puromycin | Kerafast | Cat# EQ0001, lot 041918; RRID:AB_2620162 |
| Normal Rabbit Control IgG | SinoBiological | Cat# CR1; RRID: AB_3073921 |
| Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology | Cat# 7074S; RRID:AB_2099233 |
| Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) | Abcam | Cat# ab99697, lot GR254788-1; RRID:AB_10673897 |
| Rat monoclonal [187.1] Anti-Mouse kappa light chain (HRP) | Abcam | Cat# ab99617, lot GR256873-1; RRID:AB_10673885 |
| Bacterial and virus strains | ||
| One Shot™ TOP10 Chemically Competent E. coli | Invitrogen | C404010 |
| NEB® 5-alpha Competent E. coli (High Efficiency) | New England Biolabs | C2987H |
| Chemicals, peptides, and recombinant proteins | ||
| Cycloheximide | Millipore | C4859 |
| Salmon-sperm DNA | Sigma | D7656 |
| Puromycin | Gibco | A1113803 |
| ISRIB | Sigma Aldrich | SML0843 |
| Sodium (meta)arsenite | Sigma Aldrich | S7400 |
| Trimethoprim (TMP) | Sigma Aldrich | T7883 |
| P-32 | Perkin Elmer | Phosphorus-32, as monopotassium phosphate |
| Lipofectamine transfection reagent | Invitrogen | 11668019 |
| Effectene transfection reagent | Qiagen | 301425 |
| HaloTag-MCP protein | Protein Expression and Purification facility, CSU; H. Scherman | N/A |
| JF646-HaloTag ligand | HHMI Janelia; L. Lavis | N/A |
| TURBO™ DNase | Invitrogen | AM2388 |
| SUPERase•In™ RNase Inhibitor | Invitrogen | AM2696 |
| Random Hexamers | Invitrogen | N8080127 |
| SuperScript™ IV Reverse Transcriptase | Invitrogen | 18090050 |
| Critical commercial assays | ||
| Dual-Luciferase® Reporter Assay System | Promega | E1960 |
| TRIzol™ Reagent | Invitrogen | 15596–018 |
| TRIzol™ LS Reagent | Invitrogen | 10296–010 |
| iTaq™ Universal SYBR® Green Supermix | Bio-Rad | 1725125 |
| EZview Red Protein G Affinity Gel beads | Millipore | E3403 |
| Amersham Hybond P 0.45 PVDF blotting membrane | Cytiva Life Sciences | 10600029 |
| 0.45 μM cellulose acetate Spin-X filters | Corning | CLS8162 |
| Micro Bio-Spin™ P-30 Gel Columns, Tris Buffer (RNase-free) | BioRad | 7326250 |
| ULTRAhyb-oligo hybridization buffer | Invitrogen | AM8663 |
| NuPAGE™ MES SDS Running Buffer (20X) | Invitrogen | NP0002 |
| NuPAGE™ Transfer Buffer (20X) | Invitrogen | NP00061 |
| cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail | Roche | 4693159001 |
| Qubit™ RNA Quantification, broad range kit | Invitrogen | Q10210 |
| Amersham MicroSpin S-400 HR Columns | Cytiva Life Sciences | 27514001 |
| Direct-zol RNA Micro-Prep Kit | Zymo Research | R2060 |
| Human riboPOOL for Ribosome Profiling samples | Galen Molecular | dp-K012-000042 |
| QIAseq miRNA Library Kit | Qiagen | 331502 |
| QIAseq miRNA NGS 96 Index IL plate kit | Qiagen | 331565 |
| Qubit™ dsDNA Quantitation, high sensitivity kit | Invitrogen | Q32851 |
| Hybridizing rRNA complementary DNA oligo mix | Baldwin et al., 2021132 | N/A |
| RNA Clean XP beads | Beckman Coulter | A63987 |
| KAPA RNA Hyperprep library kit | Roche | KK8540 |
| KAPA Dual-Indexed Adapter Kit | Roche | 08278555702 |
| Tapestation D1000 HS Screentape | Agilent | 5067–5584 |
| Tapestation D1000 HS Screentape reagents | Agilent | 5067–5585 |
| Deposited data | ||
| Paired Ribo-seq and RNA-seq data | This study | GEO Database: GSE202900 |
| Mendeley image dataset | This study | Mendeley Data: https://data.mendeley.com/datasets/tw2bjh857f/2 |
| Experimental models: Cell lines | ||
| HEK293T | ATCC | Cat# CRL-3216; RRID:CVCL_0063 |
| U2OS | ATCC | Cat# HTB-96; RRID:CVCL_0042 |
| Drosophila S2 | ThermoFisher | R69007 |
| ZNF598 KO Flp-In T-REx 293 dox inducible GFP-P2A-(KAAA)0-P2A-RFP | Juszkiewicz and Hegde, 201739 | N/A |
| ZNF598 KO Flp-In T-REx 293 dox inducible GFP-P2A-(KAAA)21-P2A-RFP | Juszkiewicz and Hegde, 201739 | N/A |
| eDHFR-degron tagged DDX6 HEK293T | This paper | N/A |
| Oligonucleotides | ||
| See Table S1 for full list of RT-qPCR primers, CRISPR guides, and probes | N/A | N/A |
| DNA oligonucleotide pool complementary to human rRNA transcripts | Baldwin et al., 2021132 | N/A |
| Recombinant DNA | ||
| pUB_SMFLuc0—MS2 | This paper | Addgene #210568 |
| pUB_SMFLuc100—MS2 | This paper | Addgene #210570 |
| pIS1_Opt_Renilla | This paper | Addgene 210571 |
| Nonopt_WT_Renilla_pIS1 | pIS1 | Addgene #12179 |
| pUB_SM-RLuc0—MS2 | This paper | Addgene #210572 |
| pUB_SM-RLuc100—MS2 | This paper | Addgene # 210573 |
| pUB_FLuc100—MS2 | This paper | Addgene # 210574 |
| pcDNA3.1_γHA-GFPopt | This paper | Addgene # 210575 |
| pcDNA3.1_γHA-GFPnonopt | This paper | Addgene # 210576 |
| pUB_OptγHAramp-FLuc100 | This paper | Addgene #210577 |
| pUB_NonoptγHAramp-FLuc100 | This paper | Addgene # 210579 |
| pcDNA3.1_SM-FLuc0-malat1 | This paper | Addgene # 210580 |
| pcDNA3.1_SM-FLuc100-malat1 | This paper | Addgene # 210581 |
| pcDNA3.1_SM-FLuc0-pA | This paper | Addgene # 210583 |
| pcDNA3.1_SM-FLuc100-pA | This paper | Addgene # 210584 |
| pUB_SM-FLuc0_EMCV-Ren | This paper | Addgene # 210590 |
| pUB_SM-FLuc100_EMCV-Ren | This paper | Addgene # 210591 |
| Dm_γHA-FLuc0_CSC | This paper | Addgene # 210589 |
| Dm_γHA-FLuc100_CSC | This paper | Addgene #210592 |
| Dm_Opt_Renilla_CSC | This paper | Addgene # 210593 |
| pUB_SM-FLuc0-GFPOPT | This paper | Addgene # 210594 |
| pUB_SM-FLuc100-GFPOPT | This paper | Addgene #210596 |
| pUB_mCherry-Rluc100 | This paper | Addgene # 210600 |
| lentiCRISPR v2-blast | Mohan Babu; Addgene | Addgene #83480 |
| psPax2 | Mikko Taipale | N/A |
| pCMV-VSV-G | Mikko Taipale | N/A |
| Software and algorithms | ||
| UMI-tools | Smith et al., 2017133 | N/A |
| Bowtie version 1.2.2. | Langmead et al., 2009135 | N/A |
| SAMtools version 1.12 | Danecek et al., 2021136 | N/A |
| STAR version 2.6.0a | Dobin et al., 2013137 | N/A |
| BEDtools | Quinlan and Hall, 2010141 | N/A |
| Salmon version 1.6.0. | Patro et al., 2017138 | N/A |
| Cutadapt | Martin et al., 2011134 | N/A |
| deepTools (Bamcoverage) | Ramírez et al., 2014139 | N/A |
| ribosomeProfilingQC R package | Ou and Hoye, 2022140 | N/A |
| Fiji ImageJ | Schindelin et al., 2012130 | N/A |
| Adobe Photoshop | https://www.adobe.com/products/photoshop.html | N/A |
| Adobe Illustrator | https://www.adobe.com/products/illustrator.html | N/A |
| Other | ||
| 35 mm glass bottom MatTek dishes | MatTek Corporation | P35G-1.5-14-C |
| Open-Top Polyclear Centrifuge tubes | Seton Scientific | 7030 |