(A) Schematic of DDX3X protein with human missense and nonsense mutations noted, along with helicase/RNA binding domains (red, blue). Nonsense mutations, highlighted in yellow, are predicted to act …
(A) Representative western blot of indicated genotypes probed for DDX3X (top) and β-actin loading control (bottom). Densitometric quantification of bands is shown below each lane. These data also …
Western blot anlaysis of DDX3X in embryonic brain samples.
(A) RT-qPCR quantification of Ddx3x and Ddx3y mRNA levels in FACS-isolated Tdtomato + cells from cKO male E11.5 cortices. n=5–8 embryos/condition. (B) Schematic of Ddx3y CRISPR sgRNA electroporation …
(A) Representative sections from E14.5 cortices stained with Sox2 (magenta) and Tbr2 (green) (control M and cKO M shown). (B, C) Quantification of density of Sox2+ (RGCs) (B) and Tbr2 +Sox2- (mature …
(A) Representative coronal sections from control and cHet F/cKO M at E13.5 immunostained with Sox2 (green), Tbr2 (magenta) and Hoechst (control M and cKO M shown). (B–E) Quantification of RGCs …
(A) Representative sections stained with Ki67 (green) and EdU (magenta) from E14.5 control and cHet F/cKO M mice (control F and cHet F shown) pulsed with EdU at E13.5, and higher magnification …
(A) Representative coronal sections from control and cHet F/cKO M at E14.5 immunostained with Sox2 (green), Tbr2 (red), EdU (magenta) and Hoechst (control F and cHet F shown). (B) Quantification of …
(A) Live imaging paradigm for monitoring cell fate. Figure 5A has been adapted from Figure 3A and E from Pilaz et al., 2016. (B) Live imaging DIC snapshots at indicated t=minutes or hours, and fixed …
(A) Experimental paradigm for Ribo-seq and RNAseq of E11.5 cortices from control and cKO mice. n=3/sex/condition with four embryos pooled per n. (B) TE of transcripts enriched in RGCs, IPs, deep …
(A) Representative denaturing urea gel of embryonic cortices treated with RNase I illustrating RPFs (red box). (B–D) RibosomeProfilingQC assessment of deep sequencing of cDNA libraries showing read …
Quality Control Assessment of Ribosome Profiling.
(A–C) RT-qPCR quantification of mRNA levels for Ribo-seq candidates in input samples, monosome and polysome fractions at E11.5 for Topbp1 (A) and Setd3 (B) and at E14.5 for Hax1 (C). (D) …
RNA immunoprecipitations of targets by DDX3X.
(A) RT-qPCR quantification of Setd3 knockdown in N2A cells. n=4/condition with two independent trials. (B) Representative sections of E17.5 brains from mice electroporated at E14.5 with pCAG-GFP and …
Scale bar = 300mm.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Mus musculus) | Ddx3x | NA | MGI:103064; NCBI Gene: 13,205 | |
Gene (M. musculus) | Ddx3y | NA | MGI:1349406; NCBI Gene: 26,900 | |
Gene (M. musculus) | Rcor2 | NA | MGI:1859854; NCBI Gene: 104,383 | |
Gene (M. musculus) | Setd3 | NA | MGI:1289184; NCBI Gene: 52,690 | |
Gene (M. musculus) | Topbp1 | NA | MGI:1920018; NCBI Gene: 235,559 | |
Genetic reagent (M. musculus) | C57BL/6 J | Jackson Laboratory | JAX #000664; RRID:IMSR_JAX:000664 | |
Genetic reagent (M. musculus) | Emx1-Cre | Jackson Laboratory | JAX #005628; RRID:IMSR_JAX:005628; MGI:2684610 | MGI symbol: Emx1 tm1(cre)Krj |
Genetic reagent (M. musculus) | Rosa26Ai14 | Jackson Laboratory | JAX #007914; RRID:IMSR_JAX:007914 | MGI symbol: Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze |
Genetic reagent (M. musculus) | Neurod6-Cre (NEX-Cre) | PMID:17146780 | MGI:2668659 | MGI symbol: Neurod6 tm1(cre)Kan |
Genetic reagent (M. musculus) | Ddx3xlox/lox | PMID:27179789 | MGI:5774968 | MGI symbol: Ddx3x tm1.1Lyou |
Genetic reagent (M. musculus) | Ddx3y sgRNAs | this paper | NCBI gene: 26,900 | generated with Benchling for depleting Ddx3y; see Figure 2 |
Genetic reagent (M. musculus) | smFISH probes for Ddx3x | this paper | NCBI gene: 13,205 | generated with script from Tsanov et al., 2016 for monitoring Ddx3x RNA; see Figure 1 |
Genetic reagent (M. musculus) | Scrambled siRNAs | Qiagen | Qiagen:1022076 | |
Genetic reagent (M. musculus) | Setd3 siRNAS | Qiagen | Qiagen:1027416 | Gene ID: 52,690 |
Cell line (M. musculus, male) | Neuro-2a | ATCC | ATCC:CCL-131; RRID:CVCL_0470 | |
Strain, strain background | NEB 5-alpha Competent E. coli (High Efficiency) | New England Biolabs | NEB:C2987H | |
Sequence-based reagent (M. musculus) | Rcor2 qPCR primers | Harvard PrimerBank; PMID:22086960 | Harvard PrimerBank ID: 154147710 c2; Forward 5'-TGCTTCTGTGGCATAAACACG-3'; Reverse 5'-GGCTGGGAATCACCTTGTCAG-3' | |
Sequence-based reagent (M. musculus) | Setd3 qPCR primers | Harvard PrimerBank; PMID:22086960 | Harvard PrimerBank ID: 21312266a1; Forward 5'-AAATCAGGTACTGGGGCTACA-3'; Reverse 5'-GGCCCATTTCATTAGATCAGGGA-3' | |
Sequence-based reagent (M. musculus) | Topbp1 qPCR primers | Harvard PrimerBank; PMID:22086960 | Harvard PrimerBank ID: 118130322 c1; Forward 5'-CAGGATTGTTGGTCCTCAAGTG-3'; Reverse 5'-ACAGGATACAGTTACGTCAGACA-3' | |
Antibody | anti-SOX2 (rat monoclonal) | ThermoFisher | ThermoFisher:14-9811-82; RRID:AB_11219471 | (1:1000) |
Antibody | anti-BrdU (rat monoclonal) | Abcam | Abcam:ab6326 | (1:200) |
Antibody | anti-TUJ1 (mouse monoclonal) | Biolegend | Biolegend:801202; RRID:AB_10063408 | (1:2000) |
Antibody | anti-CTIP2 (rat monoclonal) | Abcam | Abcam:AB18465 | (1:500) |
Antibody | anti-TBR2 (rabbit polyclonal) | Abcam | Abcam:AB23345; RRID:AB_778267 | (1:1000) |
Antibody | anti-CC3 (rabbit polyclonal) | Cell Signaling | Cell Signaling:9661; RRID:AB_2341188 | (1:250) |
Antibody | anti-NEUROD2 (rabbit polyclonal) | Abcam | Abcam:AB104430; RRID:AB_10975628 | (1:500) |
Antibody | anti-Ki67 (rabbit monoclonal) | Cell Signaling Technology | Cell Signaling:12,202 | (1:1000) |
Antibody | anti-PH3 (rabbit polyclonal) | Millipore | Millipore:06–570 | (1:500) |
Antibody | anti-TBR1 (rabbit monoclonal) | Cell Signaling Technology | Cell Signaling Technology:49,661 S | (1:1000) |
Antibody | anti-DDX3X (rabbit polyclonal) | Sigma Aldrich | Sigma Aldrich:HPA001648; RRID:AB_1078635 | (IF, 1:500; western, 1:1000) |
Antibody | anti-GFP (chicken polyclonal) | Abcam | Abcam:Ab13970; RRID:AB_300798 | (1:1000) |
Antibody | anti-B-actin (mouse monoclonal) | Santa Cruz | Santa Cruz:sc-47778 | (1:500) |
Antibody | anti-mouse HRP (goat polyclonal) | ThermoFisher | ThermoFisher:32430; RRID:AB_1185566 | (1:2000) |
Antibody | anti-rabbit HRP (goat polyclonal) | ThermoFisher | ThermoFisher:A16110; RRID:AB_2534782 | (1:2000) |
Antibody | anti-GFP (mouse monoclonal) | Santa Cruz | Santa Cruz:sc9996 | |
Antibody | AlexaFluor-conjugated secondary antibodies (488, 555, 568, 594, 647) | ThermoFisher | (1:500) | |
Recombinant DNA reagent | pX330-U6-Chimeric_BB-CBh-hSpCas9 | Addgene | AddGene:42230; RRID:Addgene_42230 | |
Recombinant DNA reagent | pX330-U6-Chimeric_BB-CBh-hSpCas9+Ddx3y guides | this paper | Cloned for depletion of Ddx3y; see Figure 2 | |
Recombinant DNA reagent | pCAG-GFP | PMID:32135084 | ||
Recombinant DNA reagent | pCAG-GFP-human DDX3X | PMID:32135084 | ||
Commercial assay or kit | Click-it EdU AlexaFluor 594 imaging kit | Life Technologies | Life Technologies:c10339 | |
Commercial assay or kit | Qiagen RNAeasy kit | Qiagen | Qiagen:74,034 | |
Commercial assay or kit | Qiagen miRNA library prep kit | Qiagen | Qiagen:331,502 | |
Commercial assay or kit | RiboMinus Eukaryote kit v2 | ThermoFisher | ThermoFisher:A15020 | |
Commercial assay or kit | RNA Analysis Kit (15 nt) | Agilent | Agilent:DNF-471 | |
Commercial assay or kit | Kapa mRNA HyperPrep kit with mRNA capture | KapaBiosystems | KapaBiosystems:KR1352 | |
Commercial assay or kit | iScript cDNA synthesis kit | BioRad | BioRad:1708891 | |
Commercial assay or kit | iTaq Universal Sybr Green Supermix | BioRad | BioRad:1725121 | |
Commercial assay or kit | BCA protein quantification | ThermoFisher | ThermoFisher:23,227 | |
Commercial assay or kit | ECL | ThermoFisher | ThermoFisher:32,106 | |
Chemical compound, drug | EdU | ThermoFisher | ThermoFisher:A10044 | |
Chemical compound, drug | BrdU | Sigma Aldrich | Sigma:B5002 | |
Chemical compound, drug | Cycloheximide | Calbiochem | Sigma:239,764 | |
Software, algorithm | Fiji/ImageJ | PMID:22743772 | v1.52i | |
Software, algorithm | QuPATH | PMID:29203879 | v0.3.2 | |
Software, algorithm | bowtie2 | PMID:22388286 | v2.4.4 | |
Software, algorithm | STAR | PMID:23104886 | v2.7.9a | |
Software, algorithm | RibosomeProfilingQC | DOI:10.18129/B9.bioc.ribosomeProfilingQC | v1.8.0 | |
Software, algorithm | Ribo-seQC | DOI:10.1101/601468 | v0.99 | |
Software, algorithm | DESeq2 | PMID:25516281 | v1.34.0 | |
Software, algorithm | ORFquant | PMID:33765284 | v1.02 | |
Software, algorithm | R | R Foundation for Statistical Computing | v4.1.0 | |
Other | Vectashield | Vector Labs | Vector Labs:H-1000–10 | See immunoflourescence section in methods |
Other | DAPI stain | ThermoFisher | ThermoFisher:D1306 | See FACS section in methods |
Other | Propidium iodide stain | ThermoFisher | ThermoFisher:P3566 | See FACS section in methods |
Other | Hoechst stain | ThermoFisher | ThermoFisher:H3570 | See immunoflourescence section in methods |
Other | Turbo DNase I | Invitrogen | Invitrogen:AM2238 | See RNAseq and ribosome footprinting section in methods |
Other | Superase In | Invitrogen | Invitrogen:AM2694 | See RNAseq and ribosome footprinting section in methods |
Other | TRIzol | Invitrogen | Invitrogen:15596026 | See RNAseq and ribosome footprinting section in methods |
Other | GlycoBlue | Invitrogen | Invitrogen: AM9515 | See RNAseq and ribosome footprinting section in methods |
Other | PNK enzyme | New England Biolabs | NEB:M0247S | See RNAseq and ribosome footprinting section in methods |
Other | 15% acrylamide denaturing urea-gel | BioRad | BioRad:4566053 | See RNAseq and ribosome footprinting section in methods |
Other | 2 X sample dye | Novex | Novex:LC6876 | See RNAseq and ribosome footprinting section in methods |
Other | dsDNA ladder | ThermoFisher | ThermoFisher:10488023 | See RNAseq and ribosome footprinting section in methods |
Other | miRNA ladders | New England Biolabs | NEB:N2102S | See RNAseq and ribosome footprinting section in methods |
Other | SYBR gold | Invitrogen | Invitrogen:S11494 | See RNAseq and ribosome footprinting section in methods |
Other | SpinX column | Corning | Corning:CLS8162 | See RNAseq and ribosome footprinting section in methods |
Other | TRIzol LS reagent | ThermoFisher | ThermoFisher:10296010 | See RNA Immunoprecipitation section in methods |
Other | Lipofectamine 2000 | ThermoFisher | ThermoFisher:11668019 | See RNA Immunoprecipitation section in methods |
Other | Protein G-coated Dynabeads | ThermoFisher | ThermoFisher:0003D | See RNA Immunoprecipitation section in methods |
Other | GoTaq Green Master Mix | Promega | Promega:M712 | See RNA Immunoprecipitation section in methods |
Other | 1 X RIPA buffer | Pierce | Pierce:89,900 | See SDS-PAGE and western blot analysis section in methods |
Other | 2 X sample buffer | BioRad | BioRad:1610737 | See SDS-PAGE and western blot analysis section in methods |
Other | protease inhibitors | Sigma Aldrich | Sigma:78,429 | See SDS-PAGE and western blot anlaysis section in methods |
Other | 12% polyacrylamide gel | BioRad | BioRad:4568046 | See SDS-PAGE and western blot anlaysis section in methods |
Other | PVDF membrane | BioRad | Biorad:1704157 | See SDS-PAGE and western blot anlaysis section in methods |
An excel file containing the comparison, sample size, statistical test and number of litters used (when applicable) for each data plot.
An excel file containing the primer and sgRNA sequences used in this paper.
An excel file containing three tabs (1) the differential expression analysis of controls versus Ddx3x cKO for RNAseq and Riboseq data (using only unique reads); (2) the analysis of translational efficiency from wildtype (ie: control) data (using all reads); (3) the cell type-enriched transcripts used in Figure 6B.