Journal: bioRxiv

Article Title: Functional screen for mediators of onco-mRNA translation specificity

doi: 10.1101/2024.10.10.617637

Figure Lengend Snippet: (a) Plot of the difference in dimethyl sulfate (DMS) reactivities of the MYC 5’UTR between shRBM42 versus shCNTL in Panc-1 cells. (b) DMS-Seq determined full length MYC 5’UTR RNA structure in control cells, zoom in on Stem III comparing shCNTL vs. shRBM42. Numbers indicate the average probability of the base pairing in the specified region. Red arrow indicates the predicted RBM42 bound nucleotide as show in . (c) Dual luciferase assay with the MYC 5’UTR WT or with deletion of bases 363-394, highlighted in , in Panc-1 cells. Luciferase values normalized to RNA expression levels, n=6. (d) IP followed by mass spectrometry identified endogenous RBM42 cytoplasmic protein-protein interactome in Panc-1 cells. Relevant functional groupings labeled. (e) Molecular function gene ontology enrichment of RBM42 interacting proteins. Gene ratio is the fraction of genes identified within the GO term. (f) Representative IP-western blots of eGFP-HA or RBM42-HA from the cytoplasmic fraction in the presence or absence of RNaseA treatment with 40S ribosomal subunits (RPS) and eIF2β (component of the 43S PIC). ( g ) Proximity ligation assay images and quantification of foci per cell in Panc-1 PDAC cells probing for RBM42 with either eIF2β, RPS20 or RPS3. None indicates a single primary antibody was used. Dotted lines indicate the outline of the cells, n ≥ 5 fields/condition. ( h ) Representative western blot of fractions from Panc-1 untreated or EDTA treated cytoplasmic lysate separated on a 10-50% sucrose gradient; small ribosomal subunit member, RPS6, and large ribosome subunit protein, RPL23. (i) Diagram showing the 48S pre-initiation complex formation assay. Created with BioRender.com. (j) Quantification of MYC mRNA in the free RNA versus 48S bound RNA fractions from a 10-30% sucrose gradient fractionation of control or shRBM42 knockdown Panc-1 lysate, n=4. All graphs mean ± SD. * p < 0.05, *** p <0.001, **** p <0.0001, ns = not significant (Unpaired t-test).

Article Snippet: The following antibodies were used: anti-β-Actin (A1978, Millipore Sigma), anti-UBAP2L (A300-533A, Bethyl), anti-METTL3 (A301-567A, Bethyl), anti-YTHDF2 (24744-1-AP, ProteinTech), anti-c-Myc (D84C12) (5605S, Cell Signaling), anti-RBM42 (A305-139A, Bethyl), anti-EGFR (4267, Cell Signaling), anti-c-Jun (Cell Signaling, 9165), anti-SRF (Cell Signaling, 5147), anti-PAK4 (Bethyl, A300-356A), anti-EZR (Cell Signaling, 3145), anti-RPS6 (54D2) (Cell Signaling, 2317S), anti-RPL23 (Bethyl, A305-008A), anti-puromycin (Millipore, MABE343), anti-GAPDH (Cell Signaling, 5174S), anti-Lamin A/C (Cell Signaling, 4777S), anti-LSD1 (Cell Signaling, 4064S), anti-α-tubulin (Sigma, T8203), anti-EIF2S2 (Bethyl, A301-743A), anti-RPS12 (ProteinTech, 16490-1-AP), anti-RPS3 (Cell Signaling, 9538S), anti-RPS20 (ProteinTech, 15692-1-AP), anti-RPS8 (Bethyl, A305-016A), anti-HA epitope tag (HA.11) (BioLegend, 901513).

Techniques: Control, Luciferase, RNA Expression, Mass Spectrometry, Functional Assay, Labeling, Western Blot, Proximity Ligation Assay, Tube Formation Assay, Fractionation, Knockdown

Journal: bioRxiv

Article Title: Functional screen for mediators of onco-mRNA translation specificity

doi: 10.1101/2024.10.10.617637

Figure Lengend Snippet: (a) Volcano plot showing positive control known activators and negative control genes of MYC translation from Panc-1 CRISPRi reporter screen. eIF5A was previously identified and validated as the sole hit in a MYC translational repressor screen. (b) Gene ontology molecular function enrichment for the 309 candidate repressors of MYC translation in Panc-1 cells. Gene ratio is the fraction of genes identified within the GO term. (c) mRNA expression of UBAP2L , METTL3 and YTHDF2 in healthy pancreas (GTex) versus pancreatic adenocarcinoma (PDAC) ; Welch’s t-test. (d) Protein expression of UBAP2L, METTL3 and YTHDF2 normal versus tumor from CPTAC data set; two-sided t-test . (e) Single guide validation with 2 independent sgRNAs in the Panc-1 MYC translational reporter cell line used for the CRISPRi screen at day 5 doxycycline treatment, positive controls EIF4A1 and EIF4H , n ≥ 4. (f) qPCR RNA quantification of single guide validation experiments. (g) Single guide validation of negative control genes with 2 independent sgRNAs in the Panc-1 MYC translational reporter cells, n ≥ 6. (h) qPCR RNA quantification of negative control gene experiments. (i) Quantification of RNA from UBAP2L , METTL3 and YTHDF2 knockdown experiments in Panc-1 cells. (j) qPCR analysis of MYC mRNA from 10-50% sucrose gradient fractionation of control or YTHDF2 knockdown in Panc-1 cells, n=3. (k) Total RNA quantification from UBAP2L , METTL3 and YTHDF2 knockdown polysome experiments. (l) qPCR analysis of control genes, ACTB or B2M , from 10-50% sucrose gradient fractionation of control or YTHDF2 knockdown in Panc-1 cells, n=3. (m) Correlation of UBAP2L and Myc protein abundance from human pancreatic adenocarcinoma specimens , n=17. All graphs mean ± SEM. * p < 0.05, ** p < 0.01, *** p <0.001, **** p < 0.0001, ns = not significant (total RNA quantification 1-way ANOVA, uncorrected Fisher’s LSD test relative to own shCNTL & polysome distributions 2-way ANOVA, uncorrected Fisher’s LSD test). Extended data associated with .

Article Snippet: The following antibodies were used: anti-β-Actin (A1978, Millipore Sigma), anti-UBAP2L (A300-533A, Bethyl), anti-METTL3 (A301-567A, Bethyl), anti-YTHDF2 (24744-1-AP, ProteinTech), anti-c-Myc (D84C12) (5605S, Cell Signaling), anti-RBM42 (A305-139A, Bethyl), anti-EGFR (4267, Cell Signaling), anti-c-Jun (Cell Signaling, 9165), anti-SRF (Cell Signaling, 5147), anti-PAK4 (Bethyl, A300-356A), anti-EZR (Cell Signaling, 3145), anti-RPS6 (54D2) (Cell Signaling, 2317S), anti-RPL23 (Bethyl, A305-008A), anti-puromycin (Millipore, MABE343), anti-GAPDH (Cell Signaling, 5174S), anti-Lamin A/C (Cell Signaling, 4777S), anti-LSD1 (Cell Signaling, 4064S), anti-α-tubulin (Sigma, T8203), anti-EIF2S2 (Bethyl, A301-743A), anti-RPS12 (ProteinTech, 16490-1-AP), anti-RPS3 (Cell Signaling, 9538S), anti-RPS20 (ProteinTech, 15692-1-AP), anti-RPS8 (Bethyl, A305-016A), anti-HA epitope tag (HA.11) (BioLegend, 901513).

Techniques: Positive Control, Negative Control, Expressing, Knockdown, Fractionation, Control

Journal: bioRxiv

Article Title: Functional screen for mediators of onco-mRNA translation specificity

doi: 10.1101/2024.10.10.617637

Figure Lengend Snippet: (a) Endogenous RBM42 irCLIP control RNA ligation blot -/+ UV crosslinking with titration of RNase I treatment. (b) Endogenous cytoplasmic or nuclear RBM42 CLIP-qPCR for mature MYC mRNA (lacking intron 2) fold over IgG control; n=5. Ratio paired t-test. (c) Nuclear RBM42 CLIP-qPCR positive control binding to known nuclear RNA interactions with U4 and U6 for the samples in (b); mean ± SEM. Ratio paired t-test compared to IgG control. (d) KEGG functional enrichment of genes bound in their 5’UTRs by RBM42. Terms related to cancer highlighted in red. (e) Polysome qPCR analysis of PAK4 , EZR or SRF mRNA from 10-50% sucrose gradient fractionation of control or RBM42 knockdown in Panc-1 cells; n=3, mean ± SEM. 2-way ANOVA, uncorrected Fisher’s LSD test. (f) Total RNA quantification for the RBM42 knockdown polysomes; n=3. Paired t-test. (g) Representative western blots and protein quantification for PAK4, Ezrin (EZR), and SRF with RBM42 depletion in Panc-1 cells, n=6.1-way ANOVA, uncorrected Fisher’s LSD test. (h) Dual luciferase assays (DLA) with the 5’UTRs of additional RBM42 translational target genes with control or RBM42 depletion in Panc-1 cells. Renilla (RLuc) and firefly (FLuc) luciferase values normalized to RNA expression. RNA quantification validating RBM42 depletion in the DLA samples; n=4-6, mean ± SEM. Unpaired t-tests. All graphs mean ± SD unless noted. * p < 0.05, ** p <0.01, *** p < .001, **** p < .0001, ns = not significant. Extended data associated with .

Article Snippet: The following antibodies were used: anti-β-Actin (A1978, Millipore Sigma), anti-UBAP2L (A300-533A, Bethyl), anti-METTL3 (A301-567A, Bethyl), anti-YTHDF2 (24744-1-AP, ProteinTech), anti-c-Myc (D84C12) (5605S, Cell Signaling), anti-RBM42 (A305-139A, Bethyl), anti-EGFR (4267, Cell Signaling), anti-c-Jun (Cell Signaling, 9165), anti-SRF (Cell Signaling, 5147), anti-PAK4 (Bethyl, A300-356A), anti-EZR (Cell Signaling, 3145), anti-RPS6 (54D2) (Cell Signaling, 2317S), anti-RPL23 (Bethyl, A305-008A), anti-puromycin (Millipore, MABE343), anti-GAPDH (Cell Signaling, 5174S), anti-Lamin A/C (Cell Signaling, 4777S), anti-LSD1 (Cell Signaling, 4064S), anti-α-tubulin (Sigma, T8203), anti-EIF2S2 (Bethyl, A301-743A), anti-RPS12 (ProteinTech, 16490-1-AP), anti-RPS3 (Cell Signaling, 9538S), anti-RPS20 (ProteinTech, 15692-1-AP), anti-RPS8 (Bethyl, A305-016A), anti-HA epitope tag (HA.11) (BioLegend, 901513).

Techniques: Control, Ligation, Titration, Positive Control, Binding Assay, Functional Assay, Fractionation, Knockdown, Western Blot, Luciferase, RNA Expression

Journal: bioRxiv

Article Title: Functional screen for mediators of onco-mRNA translation specificity

doi: 10.1101/2024.10.10.617637

Figure Lengend Snippet: (a) Plot of the difference in dimethyl sulfate (DMS) reactivities of the MYC 5’UTR between shRBM42 versus shCNTL in Panc-1 cells. (b) DMS-Seq determined full length MYC 5’UTR RNA structure in control cells, zoom in on Stem III comparing shCNTL vs. shRBM42. Numbers indicate the average probability of the base pairing in the specified region. Red arrow indicates the predicted RBM42 bound nucleotide as show in . (c) Dual luciferase assay with the MYC 5’UTR WT or with deletion of bases 363-394, highlighted in , in Panc-1 cells. Luciferase values normalized to RNA expression levels, n=6. (d) IP followed by mass spectrometry identified endogenous RBM42 cytoplasmic protein-protein interactome in Panc-1 cells. Relevant functional groupings labeled. (e) Molecular function gene ontology enrichment of RBM42 interacting proteins. Gene ratio is the fraction of genes identified within the GO term. (f) Representative IP-western blots of eGFP-HA or RBM42-HA from the cytoplasmic fraction in the presence or absence of RNaseA treatment with 40S ribosomal subunits (RPS) and eIF2β (component of the 43S PIC). ( g ) Proximity ligation assay images and quantification of foci per cell in Panc-1 PDAC cells probing for RBM42 with either eIF2β, RPS20 or RPS3. None indicates a single primary antibody was used. Dotted lines indicate the outline of the cells, n ≥ 5 fields/condition. ( h ) Representative western blot of fractions from Panc-1 untreated or EDTA treated cytoplasmic lysate separated on a 10-50% sucrose gradient; small ribosomal subunit member, RPS6, and large ribosome subunit protein, RPL23. (i) Diagram showing the 48S pre-initiation complex formation assay. Created with BioRender.com. (j) Quantification of MYC mRNA in the free RNA versus 48S bound RNA fractions from a 10-30% sucrose gradient fractionation of control or shRBM42 knockdown Panc-1 lysate, n=4. All graphs mean ± SD. * p < 0.05, *** p <0.001, **** p <0.0001, ns = not significant (Unpaired t-test).

Article Snippet: The following antibodies were used: anti-β-Actin (A1978, Millipore Sigma), anti-UBAP2L (A300-533A, Bethyl), anti-METTL3 (A301-567A, Bethyl), anti-YTHDF2 (24744-1-AP, ProteinTech), anti-c-Myc (D84C12) (5605S, Cell Signaling), anti-RBM42 (A305-139A, Bethyl), anti-EGFR (4267, Cell Signaling), anti-c-Jun (Cell Signaling, 9165), anti-SRF (Cell Signaling, 5147), anti-PAK4 (Bethyl, A300-356A), anti-EZR (Cell Signaling, 3145), anti-RPS6 (54D2) (Cell Signaling, 2317S), anti-RPL23 (Bethyl, A305-008A), anti-puromycin (Millipore, MABE343), anti-GAPDH (Cell Signaling, 5174S), anti-Lamin A/C (Cell Signaling, 4777S), anti-LSD1 (Cell Signaling, 4064S), anti-α-tubulin (Sigma, T8203), anti-EIF2S2 (Bethyl, A301-743A), anti-RPS12 (ProteinTech, 16490-1-AP), anti-RPS3 (Cell Signaling, 9538S), anti-RPS20 (ProteinTech, 15692-1-AP), anti-RPS8 (Bethyl, A305-016A), anti-HA epitope tag (HA.11) (BioLegend, 901513).

Techniques: Control, Luciferase, RNA Expression, Mass Spectrometry, Functional Assay, Labeling, Western Blot, Proximity Ligation Assay, Tube Formation Assay, Fractionation, Knockdown

Journal: bioRxiv

Article Title: Functional screen for mediators of onco-mRNA translation specificity

doi: 10.1101/2024.10.10.617637

Figure Lengend Snippet: (a) mRNA expression of RBM42 in healthy pancreas (GTex) versus pancreatic adenocarcinoma (PDAC) ; Welch’s T-test. (b) Protein expression of RBM42 in normal pancreas versus PDAC tissues from CPTAC data set ; two-sided t-test. (c) Overall or disease-free survival correlation with RBM42 RNA expression in PDAC , . (d) Western blot analysis and quantification of Myc protein expression with RBM42 knockdown in a panel of pancreatic cancer cell lines. Bar graphs n=3-5; mean ± SD. (e) Representative western blot of puromycin incorporation assay of global translation with RBM42 depletion in Panc-1 cells. Values indicate normalized puromycin incorporation relative to shCNTL. (f) Representative polysome profiles for control or RBM42 knockdown in Panc-1 cells. Inset shows quantification of total RBM42 and MYC mRNA for the polysome profiling experiments in (g), mean ± SD. (g) qPCR analysis of MYC or control TUBB mRNA from 10-50% sucrose gradient fractionation of control or RBM42 knockdown in Panc-1 cells; n=3; mean ± SD. (h) RBM42 immunohistochemistry (IHC) on human PDAC tissue. Patient 1: grade 1, stage IIB; Patient 2: grade 2, stage not reported; Patient 3: grade 2, stage IV; Patient 4: grade 2, stage not reported. Scale bar = 50μm. ( i ) RBM42 IHC staining of mouse tissue and quantification of the cytoplasmic/nuclear RBM42 staining. Normal = Pft1a-Cre +/- , PanIN = Ptf1a-Cre +/- ;Kras G12D/+ , and PDAC = Ptf1a-Cre +/- ;Kras G12D/+ ;Trp53 flox/+ ; n = 3 mice/condition, 3 fields/animal; mean ± SD. Unpaired t-test. Scale bar = 50μm. * p < 0.05, ** p < 0.01, *** p <0.001, **** p < .0001, ns = not significant (1- or 2-way ANOVA, uncorrected Fisher’s LSD test).

Article Snippet: The following antibodies were used: anti-β-Actin (A1978, Millipore Sigma), anti-UBAP2L (A300-533A, Bethyl), anti-METTL3 (A301-567A, Bethyl), anti-YTHDF2 (24744-1-AP, ProteinTech), anti-c-Myc (D84C12) (5605S, Cell Signaling), anti-RBM42 (A305-139A, Bethyl), anti-EGFR (4267, Cell Signaling), anti-c-Jun (Cell Signaling, 9165), anti-SRF (Cell Signaling, 5147), anti-PAK4 (Bethyl, A300-356A), anti-EZR (Cell Signaling, 3145), anti-RPS6 (54D2) (Cell Signaling, 2317S), anti-RPL23 (Bethyl, A305-008A), anti-puromycin (Millipore, MABE343), anti-GAPDH (Cell Signaling, 5174S), anti-Lamin A/C (Cell Signaling, 4777S), anti-LSD1 (Cell Signaling, 4064S), anti-α-tubulin (Sigma, T8203), anti-EIF2S2 (Bethyl, A301-743A), anti-RPS12 (ProteinTech, 16490-1-AP), anti-RPS3 (Cell Signaling, 9538S), anti-RPS20 (ProteinTech, 15692-1-AP), anti-RPS8 (Bethyl, A305-016A), anti-HA epitope tag (HA.11) (BioLegend, 901513).

Techniques: Expressing, RNA Expression, Western Blot, Knockdown, Control, Fractionation, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: Functional screen for mediators of onco-mRNA translation specificity

doi: 10.1101/2024.10.10.617637

Figure Lengend Snippet: (a) Western blot analysis and quantification of RNA and protein expression of Myc and RBM42 with RBM42 knockdown in MiaPaca2 pancreatic cancer cells, n=3. (b) qPCR RNA quantification for the knockdown experiments in . PDAC cell line RNA and protein quantification 1-way ANOVA, uncorrected Fisher’s LSD test relative to own shCNTL. (c) Enriched Hallmarks and transcription factor (TF) targets from gene set enrichment analysis (GSEA) of genes with altered expression in Panc-1 shCNTL vs. shRBM42 RNA-seq data sets, Normalized Enrichment Score (NES). ( d ) qPCR quantification of RNA expression of core Myc targets and previously reported Myc targets in PDAC (blue text) in Panc-1 cells with MYC or RBM42 depletion; mean ± SEM. Unpaired t-test relative to shCNTL. ( e ) Western blot analysis and quantification of RNA and protein expression of Myc and RBM42 with MYC knockdown in Panc-1 pancreatic cancer cells, n=3. Unpaired t-test. ( f ) Quantification and representative western blots of RBM42 and Myc RNA and protein levels with exogenous Myc expression in HPDE cells, n=4. Unpaired Welch’s t-test. (g) Quantification and representative western blots of RBM42 and Myc RNA and protein levels with exogenous RBM42 expression in HPDE cells, n=4. Unpaired Welch’s t-test. (h) Representative western blots of puromycin incorporation assay of global translation with RBM42 depletion in additional pancreatic cancer cell lines. Values indicate normalized puromycin incorporation relative to shCNTL. * p < 0.05, ** p < .01, *** p < 0.001, **** p < 0.0001, and ns = not significant. All graphs are mean ± SD unless otherwise indicated. Extended data associated with .

Article Snippet: The following antibodies were used: anti-β-Actin (A1978, Millipore Sigma), anti-UBAP2L (A300-533A, Bethyl), anti-METTL3 (A301-567A, Bethyl), anti-YTHDF2 (24744-1-AP, ProteinTech), anti-c-Myc (D84C12) (5605S, Cell Signaling), anti-RBM42 (A305-139A, Bethyl), anti-EGFR (4267, Cell Signaling), anti-c-Jun (Cell Signaling, 9165), anti-SRF (Cell Signaling, 5147), anti-PAK4 (Bethyl, A300-356A), anti-EZR (Cell Signaling, 3145), anti-RPS6 (54D2) (Cell Signaling, 2317S), anti-RPL23 (Bethyl, A305-008A), anti-puromycin (Millipore, MABE343), anti-GAPDH (Cell Signaling, 5174S), anti-Lamin A/C (Cell Signaling, 4777S), anti-LSD1 (Cell Signaling, 4064S), anti-α-tubulin (Sigma, T8203), anti-EIF2S2 (Bethyl, A301-743A), anti-RPS12 (ProteinTech, 16490-1-AP), anti-RPS3 (Cell Signaling, 9538S), anti-RPS20 (ProteinTech, 15692-1-AP), anti-RPS8 (Bethyl, A305-016A), anti-HA epitope tag (HA.11) (BioLegend, 901513).

Techniques: Western Blot, Expressing, Knockdown, RNA Sequencing Assay, RNA Expression

Journal: bioRxiv

Article Title: Functional screen for mediators of onco-mRNA translation specificity

doi: 10.1101/2024.10.10.617637

Figure Lengend Snippet: (a) Myc protein cycloheximide (CHX) stability assay quantification and representative western blots in Panc-1 cells, n=2. Both bands were quantified for Myc protein levels. P-value of the difference between the slopes. Values under blot indicate normalized Myc protein expression relative to shCNTL; mean ± SD. (b) qPCR quantification of retained introns for MYC and control mRNA B2M , n=3. 2-way ANOVA, uncorrected Fisher’s LSD test. (c) Panc-1 shCNTL and shRBM42 RNA-seq global splicing analysis (rMATS) collapsed to gene level, graphing the event with the highest DPSI, (n=3 replicates/condition). Table events FDR < 0.05 and |DPSI| > 0.1. ( d ) Additional human patient PDAC specimen RBM42 immunohistochemistry (IHC). Disease grade (G) and stage, where reported, are listed. Scale bar = 50μm. (e) Representative western blots of cytoplasmic and nuclear fractionation of pancreatic cancer cell lines. Graphs are mean ± SEM unless otherwise noted. ns = not significant. Extended data associated with .

Article Snippet: The following antibodies were used: anti-β-Actin (A1978, Millipore Sigma), anti-UBAP2L (A300-533A, Bethyl), anti-METTL3 (A301-567A, Bethyl), anti-YTHDF2 (24744-1-AP, ProteinTech), anti-c-Myc (D84C12) (5605S, Cell Signaling), anti-RBM42 (A305-139A, Bethyl), anti-EGFR (4267, Cell Signaling), anti-c-Jun (Cell Signaling, 9165), anti-SRF (Cell Signaling, 5147), anti-PAK4 (Bethyl, A300-356A), anti-EZR (Cell Signaling, 3145), anti-RPS6 (54D2) (Cell Signaling, 2317S), anti-RPL23 (Bethyl, A305-008A), anti-puromycin (Millipore, MABE343), anti-GAPDH (Cell Signaling, 5174S), anti-Lamin A/C (Cell Signaling, 4777S), anti-LSD1 (Cell Signaling, 4064S), anti-α-tubulin (Sigma, T8203), anti-EIF2S2 (Bethyl, A301-743A), anti-RPS12 (ProteinTech, 16490-1-AP), anti-RPS3 (Cell Signaling, 9538S), anti-RPS20 (ProteinTech, 15692-1-AP), anti-RPS8 (Bethyl, A305-016A), anti-HA epitope tag (HA.11) (BioLegend, 901513).

Techniques: Stability Assay, Western Blot, Expressing, Control, RNA Sequencing Assay, Immunohistochemistry, Fractionation

Journal: bioRxiv

Article Title: Functional screen for mediators of onco-mRNA translation specificity

doi: 10.1101/2024.10.10.617637

Figure Lengend Snippet: (a) Pancreatic cancer cell line viability or cell growth time course with control (CNTL) or RBM42 shRNA knockdown using Cell Titer Glo Assay normalized to day 0 n= 6 (Panc-1 & MiPaca2) or 9 (BxPC3) or total live cell counting (trypan blue negative) relative to the number of cells seeded (n = 4-6 replicates). Unpaired t-test. (b) Two-dimensional colony formation with control or RBM42 depletion stained with crystal violet; n=10-12. (c) Anchorage independent growth in soft agar with control or shRBM42 stained with MTT to visualize live colonies; n=4-6. (d) Myc coding sequence (CDS) rescue of shRBM42 induced loss of PDAC cell viability. To calculate the cell viability relative to EV + shRBM42, all readings were normalized to their day 0 values to account for cell seeding. Then, for each timepoint, all conditions were compared to the average of knockdown (EV + shRBM42) viability readings for that day to calculate the fold rescue.; n=10 (BxPC3) or 6 (MiaPaca2), significance at day 4 mean ± SEM. Diagram created with BioRender.com. (e) Representative western blot of the Myc and RBM42 expression for cell viability rescue experiments. Graphs are mean ± SD unless noted. Ordinary 1-way ANOVA with Dunnett’s Multiple Comparison Test unless otherwise indicated. * p < 0.05, ** p < 0.01, *** p <0.001, **** p < .0001, ns = not significant.

Article Snippet: The following antibodies were used: anti-β-Actin (A1978, Millipore Sigma), anti-UBAP2L (A300-533A, Bethyl), anti-METTL3 (A301-567A, Bethyl), anti-YTHDF2 (24744-1-AP, ProteinTech), anti-c-Myc (D84C12) (5605S, Cell Signaling), anti-RBM42 (A305-139A, Bethyl), anti-EGFR (4267, Cell Signaling), anti-c-Jun (Cell Signaling, 9165), anti-SRF (Cell Signaling, 5147), anti-PAK4 (Bethyl, A300-356A), anti-EZR (Cell Signaling, 3145), anti-RPS6 (54D2) (Cell Signaling, 2317S), anti-RPL23 (Bethyl, A305-008A), anti-puromycin (Millipore, MABE343), anti-GAPDH (Cell Signaling, 5174S), anti-Lamin A/C (Cell Signaling, 4777S), anti-LSD1 (Cell Signaling, 4064S), anti-α-tubulin (Sigma, T8203), anti-EIF2S2 (Bethyl, A301-743A), anti-RPS12 (ProteinTech, 16490-1-AP), anti-RPS3 (Cell Signaling, 9538S), anti-RPS20 (ProteinTech, 15692-1-AP), anti-RPS8 (Bethyl, A305-016A), anti-HA epitope tag (HA.11) (BioLegend, 901513).

Techniques: Control, shRNA, Knockdown, Glo Assay, Cell Counting, Staining, Sequencing, Western Blot, Expressing, Comparison

Journal: bioRxiv

Article Title: Functional screen for mediators of onco-mRNA translation specificity

doi: 10.1101/2024.10.10.617637

Figure Lengend Snippet: (a) Myc protein and RNA expression levels from the experiments in . (b) RBM42 protein and RNA expression levels from the experiments in . Graphs are mean ± SD; 1-way ANOVA, uncorrected Fisher’s LSD test relative to own shCNTL. (c) Representative western blots demonstrating for the PDAC cell line growth with RBM42 depletion. Graphs are mean ± SEM; unpaired t-test relative to shCNTL. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant. Extended data associated with .

Article Snippet: The following antibodies were used: anti-β-Actin (A1978, Millipore Sigma), anti-UBAP2L (A300-533A, Bethyl), anti-METTL3 (A301-567A, Bethyl), anti-YTHDF2 (24744-1-AP, ProteinTech), anti-c-Myc (D84C12) (5605S, Cell Signaling), anti-RBM42 (A305-139A, Bethyl), anti-EGFR (4267, Cell Signaling), anti-c-Jun (Cell Signaling, 9165), anti-SRF (Cell Signaling, 5147), anti-PAK4 (Bethyl, A300-356A), anti-EZR (Cell Signaling, 3145), anti-RPS6 (54D2) (Cell Signaling, 2317S), anti-RPL23 (Bethyl, A305-008A), anti-puromycin (Millipore, MABE343), anti-GAPDH (Cell Signaling, 5174S), anti-Lamin A/C (Cell Signaling, 4777S), anti-LSD1 (Cell Signaling, 4064S), anti-α-tubulin (Sigma, T8203), anti-EIF2S2 (Bethyl, A301-743A), anti-RPS12 (ProteinTech, 16490-1-AP), anti-RPS3 (Cell Signaling, 9538S), anti-RPS20 (ProteinTech, 15692-1-AP), anti-RPS8 (Bethyl, A305-016A), anti-HA epitope tag (HA.11) (BioLegend, 901513).

Techniques: RNA Expression, Western Blot

Journal: bioRxiv

Article Title: Functional screen for mediators of onco-mRNA translation specificity

doi: 10.1101/2024.10.10.617637

Figure Lengend Snippet: (a) Scheme of the endogenous cytoplasmic RBM42 CLIP-seq workflow in Panc-1 cells. Created with BioRender.com. (b) CLIP reads and crosslinked induced mutation sites (CIMS) on the MYC mRNA, inset of 5’UTR (CIMS FDR < 0.05). (c) Diagram of polysome sequencing and translational efficiency calculation in Panc-1 cells. (d) Volcano plot of translational efficiency of genes bound by RBM42 in the 5’UTR, key pro-oncogenic genes highlighted. (e) Venn diagram of RBM42 5’UTR bound CLIP genes (FDR < .05) and RBM42 translationally downregulated genes (adj p-value < 0.05 and FC < 0.75 [log 2 FC < -0.42]). (f) Functional enrichment of Elsevier Pathways of CLIP/Poly-Seq overlapping genes. (g) CLIP tracks for additional RBM42 bound and translationally regulated targets, EGFR and JUN . Arrow indicates the orientation of the gene, inset of 5’UTR (CIMS FDR < 0.05) (h) qPCR analysis of EGFR or JUN mRNA from 10-50% sucrose gradient fractionation of control or RBM42 knockdown in Panc-1 cells; n=3; mean ± SEM. (2-way ANOVA, uncorrected Fisher’s LSD test). (i) Representative western blots of EGFR and c-Jun protein expression with RBM42 depletion. (j) Protein quantification for EGFR and c-Jun with RBM42 depletion in Panc-1 cells (n = 6), unpaired t-test. All graphs mean ± SD unless noted. * p < 0.05, ** p <0.01, **** p < .0001.

Article Snippet: The following antibodies were used: anti-β-Actin (A1978, Millipore Sigma), anti-UBAP2L (A300-533A, Bethyl), anti-METTL3 (A301-567A, Bethyl), anti-YTHDF2 (24744-1-AP, ProteinTech), anti-c-Myc (D84C12) (5605S, Cell Signaling), anti-RBM42 (A305-139A, Bethyl), anti-EGFR (4267, Cell Signaling), anti-c-Jun (Cell Signaling, 9165), anti-SRF (Cell Signaling, 5147), anti-PAK4 (Bethyl, A300-356A), anti-EZR (Cell Signaling, 3145), anti-RPS6 (54D2) (Cell Signaling, 2317S), anti-RPL23 (Bethyl, A305-008A), anti-puromycin (Millipore, MABE343), anti-GAPDH (Cell Signaling, 5174S), anti-Lamin A/C (Cell Signaling, 4777S), anti-LSD1 (Cell Signaling, 4064S), anti-α-tubulin (Sigma, T8203), anti-EIF2S2 (Bethyl, A301-743A), anti-RPS12 (ProteinTech, 16490-1-AP), anti-RPS3 (Cell Signaling, 9538S), anti-RPS20 (ProteinTech, 15692-1-AP), anti-RPS8 (Bethyl, A305-016A), anti-HA epitope tag (HA.11) (BioLegend, 901513).

Techniques: Mutagenesis, Sequencing, Functional Assay, Fractionation, Control, Knockdown, Western Blot, Expressing

Journal: bioRxiv

Article Title: Functional screen for mediators of onco-mRNA translation specificity

doi: 10.1101/2024.10.10.617637

Figure Lengend Snippet: (a) Endogenous RBM42 irCLIP control RNA ligation blot -/+ UV crosslinking with titration of RNase I treatment. (b) Endogenous cytoplasmic or nuclear RBM42 CLIP-qPCR for mature MYC mRNA (lacking intron 2) fold over IgG control; n=5. Ratio paired t-test. (c) Nuclear RBM42 CLIP-qPCR positive control binding to known nuclear RNA interactions with U4 and U6 for the samples in (b); mean ± SEM. Ratio paired t-test compared to IgG control. (d) KEGG functional enrichment of genes bound in their 5’UTRs by RBM42. Terms related to cancer highlighted in red. (e) Polysome qPCR analysis of PAK4 , EZR or SRF mRNA from 10-50% sucrose gradient fractionation of control or RBM42 knockdown in Panc-1 cells; n=3, mean ± SEM. 2-way ANOVA, uncorrected Fisher’s LSD test. (f) Total RNA quantification for the RBM42 knockdown polysomes; n=3. Paired t-test. (g) Representative western blots and protein quantification for PAK4, Ezrin (EZR), and SRF with RBM42 depletion in Panc-1 cells, n=6.1-way ANOVA, uncorrected Fisher’s LSD test. (h) Dual luciferase assays (DLA) with the 5’UTRs of additional RBM42 translational target genes with control or RBM42 depletion in Panc-1 cells. Renilla (RLuc) and firefly (FLuc) luciferase values normalized to RNA expression. RNA quantification validating RBM42 depletion in the DLA samples; n=4-6, mean ± SEM. Unpaired t-tests. All graphs mean ± SD unless noted. * p < 0.05, ** p <0.01, *** p < .001, **** p < .0001, ns = not significant. Extended data associated with .

Article Snippet: The following antibodies were used: anti-β-Actin (A1978, Millipore Sigma), anti-UBAP2L (A300-533A, Bethyl), anti-METTL3 (A301-567A, Bethyl), anti-YTHDF2 (24744-1-AP, ProteinTech), anti-c-Myc (D84C12) (5605S, Cell Signaling), anti-RBM42 (A305-139A, Bethyl), anti-EGFR (4267, Cell Signaling), anti-c-Jun (Cell Signaling, 9165), anti-SRF (Cell Signaling, 5147), anti-PAK4 (Bethyl, A300-356A), anti-EZR (Cell Signaling, 3145), anti-RPS6 (54D2) (Cell Signaling, 2317S), anti-RPL23 (Bethyl, A305-008A), anti-puromycin (Millipore, MABE343), anti-GAPDH (Cell Signaling, 5174S), anti-Lamin A/C (Cell Signaling, 4777S), anti-LSD1 (Cell Signaling, 4064S), anti-α-tubulin (Sigma, T8203), anti-EIF2S2 (Bethyl, A301-743A), anti-RPS12 (ProteinTech, 16490-1-AP), anti-RPS3 (Cell Signaling, 9538S), anti-RPS20 (ProteinTech, 15692-1-AP), anti-RPS8 (Bethyl, A305-016A), anti-HA epitope tag (HA.11) (BioLegend, 901513).

Techniques: Control, Ligation, Titration, Positive Control, Binding Assay, Functional Assay, Fractionation, Knockdown, Western Blot, Luciferase, RNA Expression

Journal: bioRxiv

Article Title: Functional screen for mediators of onco-mRNA translation specificity

doi: 10.1101/2024.10.10.617637

Figure Lengend Snippet: (a) Plot of the difference in dimethyl sulfate (DMS) reactivities of the MYC 5’UTR between shRBM42 versus shCNTL in Panc-1 cells. (b) DMS-Seq determined full length MYC 5’UTR RNA structure in control cells, zoom in on Stem III comparing shCNTL vs. shRBM42. Numbers indicate the average probability of the base pairing in the specified region. Red arrow indicates the predicted RBM42 bound nucleotide as show in . (c) Dual luciferase assay with the MYC 5’UTR WT or with deletion of bases 363-394, highlighted in , in Panc-1 cells. Luciferase values normalized to RNA expression levels, n=6. (d) IP followed by mass spectrometry identified endogenous RBM42 cytoplasmic protein-protein interactome in Panc-1 cells. Relevant functional groupings labeled. (e) Molecular function gene ontology enrichment of RBM42 interacting proteins. Gene ratio is the fraction of genes identified within the GO term. (f) Representative IP-western blots of eGFP-HA or RBM42-HA from the cytoplasmic fraction in the presence or absence of RNaseA treatment with 40S ribosomal subunits (RPS) and eIF2β (component of the 43S PIC). ( g ) Proximity ligation assay images and quantification of foci per cell in Panc-1 PDAC cells probing for RBM42 with either eIF2β, RPS20 or RPS3. None indicates a single primary antibody was used. Dotted lines indicate the outline of the cells, n ≥ 5 fields/condition. ( h ) Representative western blot of fractions from Panc-1 untreated or EDTA treated cytoplasmic lysate separated on a 10-50% sucrose gradient; small ribosomal subunit member, RPS6, and large ribosome subunit protein, RPL23. (i) Diagram showing the 48S pre-initiation complex formation assay. Created with BioRender.com. (j) Quantification of MYC mRNA in the free RNA versus 48S bound RNA fractions from a 10-30% sucrose gradient fractionation of control or shRBM42 knockdown Panc-1 lysate, n=4. All graphs mean ± SD. * p < 0.05, *** p <0.001, **** p <0.0001, ns = not significant (Unpaired t-test).

Article Snippet: The following antibodies were used: anti-β-Actin (A1978, Millipore Sigma), anti-UBAP2L (A300-533A, Bethyl), anti-METTL3 (A301-567A, Bethyl), anti-YTHDF2 (24744-1-AP, ProteinTech), anti-c-Myc (D84C12) (5605S, Cell Signaling), anti-RBM42 (A305-139A, Bethyl), anti-EGFR (4267, Cell Signaling), anti-c-Jun (Cell Signaling, 9165), anti-SRF (Cell Signaling, 5147), anti-PAK4 (Bethyl, A300-356A), anti-EZR (Cell Signaling, 3145), anti-RPS6 (54D2) (Cell Signaling, 2317S), anti-RPL23 (Bethyl, A305-008A), anti-puromycin (Millipore, MABE343), anti-GAPDH (Cell Signaling, 5174S), anti-Lamin A/C (Cell Signaling, 4777S), anti-LSD1 (Cell Signaling, 4064S), anti-α-tubulin (Sigma, T8203), anti-EIF2S2 (Bethyl, A301-743A), anti-RPS12 (ProteinTech, 16490-1-AP), anti-RPS3 (Cell Signaling, 9538S), anti-RPS20 (ProteinTech, 15692-1-AP), anti-RPS8 (Bethyl, A305-016A), anti-HA epitope tag (HA.11) (BioLegend, 901513).

Techniques: Control, Luciferase, RNA Expression, Mass Spectrometry, Functional Assay, Labeling, Western Blot, Proximity Ligation Assay, Tube Formation Assay, Fractionation, Knockdown

Journal: bioRxiv

Article Title: Functional screen for mediators of onco-mRNA translation specificity

doi: 10.1101/2024.10.10.617637

Figure Lengend Snippet: (a) DMS-Seq determined full length MYC 5’UTR RNA structure in Panc-1 cells with RBM42 depletion. Red arrow indicates the RBM42 binding site. Box shows compensatory mutations red = 5’ mutations and blue = 3’ compensatory mutations to maintain structure. ( b ) MYC 5’UTR Stem III from shRBM42 structure showing the changes in DMS reactivity shRBM42 relative to shCNTL. (c) qPCR quantification of RBM42 mRNA levels in the dual luciferase assay experiments in and ED (1-way ANOVA, uncorrected Fisher’s LSD test to WT shCNTL). ( e ) DMS-Seq determined full length EGFR 5’UTR RNA structure in Panc-1 cells. Red arrow indicates the RBM42 binding site. ( f ) DMS-Seq determined full length JUN 5’UTR RNA structure in Panc-1 cells. Red arrows indicate the RBM42 binding sites. (g) Cytoplasmic and nuclear fraction blot to confirm proper fractionation for co-IP blots in . Total RNA gel to confirm RNaseA digestion. (h) Representative IP-western blots of eGFP-HA or RBM42-HA from the nuclear fraction confirming known RBM42 interaction with SNRNP200 and PRPF8, but lack of interaction with 40S ribosomal components [corresponding fractionation blot in (g)]. (i) Representative IP-western blots of eGFP-HA or RBM42-HA from the cytoplasmic fraction probing for IP negative controls (EGFR, HSP90 and GAPDH) and initiation factors previously implicated in MYC translation initiation (eIF4A1, eIF5A and eIF4E). (j) 48S PIC assembly assay with control mRNA TUBB and quantification of decreased RBM42 and Myc protein levels for the experiment in (Unpaired t-tests). ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant. Extended data associated with .

Article Snippet: The following antibodies were used: anti-β-Actin (A1978, Millipore Sigma), anti-UBAP2L (A300-533A, Bethyl), anti-METTL3 (A301-567A, Bethyl), anti-YTHDF2 (24744-1-AP, ProteinTech), anti-c-Myc (D84C12) (5605S, Cell Signaling), anti-RBM42 (A305-139A, Bethyl), anti-EGFR (4267, Cell Signaling), anti-c-Jun (Cell Signaling, 9165), anti-SRF (Cell Signaling, 5147), anti-PAK4 (Bethyl, A300-356A), anti-EZR (Cell Signaling, 3145), anti-RPS6 (54D2) (Cell Signaling, 2317S), anti-RPL23 (Bethyl, A305-008A), anti-puromycin (Millipore, MABE343), anti-GAPDH (Cell Signaling, 5174S), anti-Lamin A/C (Cell Signaling, 4777S), anti-LSD1 (Cell Signaling, 4064S), anti-α-tubulin (Sigma, T8203), anti-EIF2S2 (Bethyl, A301-743A), anti-RPS12 (ProteinTech, 16490-1-AP), anti-RPS3 (Cell Signaling, 9538S), anti-RPS20 (ProteinTech, 15692-1-AP), anti-RPS8 (Bethyl, A305-016A), anti-HA epitope tag (HA.11) (BioLegend, 901513).

Techniques: Binding Assay, Luciferase, Fractionation, Co-Immunoprecipitation Assay, Western Blot, Control

Journal: bioRxiv

Article Title: Functional screen for mediators of onco-mRNA translation specificity

doi: 10.1101/2024.10.10.617637

Figure Lengend Snippet: (a) Correlation of RBM42 and Myc protein abundance from human pancreatic adenocarcinoma specimens , n=16. Graph and statistics generated with cBio Cancer Genomics Portal . (b) Kaplan-Meier survival plot comparing patients in (a) with high Myc+RBM42 compared to low Myc+RBM42 protein levels. (c) Correlation of RBM42 and MYC mRNA abundance in the same human pancreatic adenocarcinoma specimens as in (a). (d) MYC Targets gene set enrichment analysis (GSEA) comparing top quartile vs. bottom quartile of RBM42 RNA expression in TGCA PAAD RNA-sequencing data (n=45 patients/quartile), Normalized Enrichment Score (NES). (e) Representative Myc and RBM42 immunofluorescence co-staining in human PDAC tissue and quantification of RBM42 and Myc per cell overlap/patient; n = 6-49 ducts/patient, mean ± SEM. Scale bar = 5μm. (f) Tumor volumes of MiaPaca2 pancreatic cancer cell xenografts with control and RBM42 knockdown, n=7 mice/group. (g) Tumor volumes of BxPC3 pancreatic cancer cell xenografts with control, RBM42 depletion or RBM42 depletion with enforced Myc expression (coding sequence), n= 8-12 mice/group. (i) Final tumor weights of BxPC3 xenografts. (j) Model of RBM42 selective translational control of pro-tumorigenic mRNAs, as exemplified by MYC . All graphs mean ± SEM unless noted. * p < 0.05, ** p < 0.01, *** p <0.001, **** p <0.0001 (Unpaired t-test).

Article Snippet: The following antibodies were used: anti-β-Actin (A1978, Millipore Sigma), anti-UBAP2L (A300-533A, Bethyl), anti-METTL3 (A301-567A, Bethyl), anti-YTHDF2 (24744-1-AP, ProteinTech), anti-c-Myc (D84C12) (5605S, Cell Signaling), anti-RBM42 (A305-139A, Bethyl), anti-EGFR (4267, Cell Signaling), anti-c-Jun (Cell Signaling, 9165), anti-SRF (Cell Signaling, 5147), anti-PAK4 (Bethyl, A300-356A), anti-EZR (Cell Signaling, 3145), anti-RPS6 (54D2) (Cell Signaling, 2317S), anti-RPL23 (Bethyl, A305-008A), anti-puromycin (Millipore, MABE343), anti-GAPDH (Cell Signaling, 5174S), anti-Lamin A/C (Cell Signaling, 4777S), anti-LSD1 (Cell Signaling, 4064S), anti-α-tubulin (Sigma, T8203), anti-EIF2S2 (Bethyl, A301-743A), anti-RPS12 (ProteinTech, 16490-1-AP), anti-RPS3 (Cell Signaling, 9538S), anti-RPS20 (ProteinTech, 15692-1-AP), anti-RPS8 (Bethyl, A305-016A), anti-HA epitope tag (HA.11) (BioLegend, 901513).

Techniques: Generated, RNA Expression, RNA Sequencing Assay, Immunofluorescence, Staining, Control, Knockdown, Expressing, Sequencing

Journal: bioRxiv

Article Title: Functional screen for mediators of onco-mRNA translation specificity

doi: 10.1101/2024.10.10.617637

Figure Lengend Snippet: (a) Gene set enrichment analysis (GSEA) top 5 hallmarks comparing top quartile vs. bottom quartile of RBM42 RNA expression in TGCA PAAD RNA-sequencing data (n=45 patients/quartile), Normalized Enrichment Score (NES). (b) Additional representative Myc and RBM42 immunofluorescence co-staining in human PDAC tissue. Location of higher magnification images shown in indicated. Higer magnification images scale bar = 5μm. (c) Quantification of RBM42 low protein expression and Myc expression per cell overlap/patient; n = 6-48 ducts/patient, mean ± SEM. (d) MiaPaca2 and (e) BxPC3 Myc and RBM42 protein expression levels in the cells injected into subcutaneously into mice. Extended data associated with .

Article Snippet: The following antibodies were used: anti-β-Actin (A1978, Millipore Sigma), anti-UBAP2L (A300-533A, Bethyl), anti-METTL3 (A301-567A, Bethyl), anti-YTHDF2 (24744-1-AP, ProteinTech), anti-c-Myc (D84C12) (5605S, Cell Signaling), anti-RBM42 (A305-139A, Bethyl), anti-EGFR (4267, Cell Signaling), anti-c-Jun (Cell Signaling, 9165), anti-SRF (Cell Signaling, 5147), anti-PAK4 (Bethyl, A300-356A), anti-EZR (Cell Signaling, 3145), anti-RPS6 (54D2) (Cell Signaling, 2317S), anti-RPL23 (Bethyl, A305-008A), anti-puromycin (Millipore, MABE343), anti-GAPDH (Cell Signaling, 5174S), anti-Lamin A/C (Cell Signaling, 4777S), anti-LSD1 (Cell Signaling, 4064S), anti-α-tubulin (Sigma, T8203), anti-EIF2S2 (Bethyl, A301-743A), anti-RPS12 (ProteinTech, 16490-1-AP), anti-RPS3 (Cell Signaling, 9538S), anti-RPS20 (ProteinTech, 15692-1-AP), anti-RPS8 (Bethyl, A305-016A), anti-HA epitope tag (HA.11) (BioLegend, 901513).

Techniques: RNA Expression, RNA Sequencing Assay, Immunofluorescence, Staining, Expressing, Injection