anti pum1  (Bethyl)

Journal: bioRxiv

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to RNA-binding protein-directed decay

doi: 10.1101/2025.10.02.680050

Figure Lengend Snippet: A. PUM1&2 repression activity was measured in HCT116 cells using Nano-luciferase reporters with three PREs within a minimal 3′ UTR with cleavage and poly-adenylation signals (Nluc 3xPRE), calculated relative to a version wherein the PRE sequences were mutated (indicated in red text) to prevent PUM binding (Nluc 3xPREmt). PUM repression of the poly-adenylated reporter was compared to a Nluc reporter that has a 3′ end generated by the MALAT1 non-coding RNA (Nluc 3xPRE MALAT), which is processed by RNase P mediated cleavage to form a triple helix structure (PBD: 4PLX). Derivatives of the Nluc 3xPRE were constructed with internal poly(A) tracts of either 20 (A20) or 60 (A60) adenosines, inserted between the PREs and the MALAT1 triple helix. Firefly luciferase (Fluc) served as an internal control to normalize transfection efficiency. B. PRE dependent repression by endogenous PUM1 and PUM2 was measured as log fold change (FC) of each Nluc 3xPRE reporter relative to its corresponding 3xPREmt reporter. Mean fold change is plotted along with individual replicate data points. n=9; 3 experiments, each with 3 biological replicates; +/- standard deviation (SD). For significance calling, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = **** based on ordinary one-way ANOVA and Tukey test for multiple comparisons. Asterisks above the axis denote significance relative to the 3xPREmt version of each reporter type, whereas below the bars are calculated relative to the poly-adenylated Nluc reporters. C. Western blot confirming the depletion of PAN2 and CNOT1 proteins by RNAi in HCT116 cells. GAPDH served as a loading control. n=3 experimental replicates. D. The effect of CCR4-NOT or PAN2 and PAN3 knockdown on PUM repression of the Nluc 3xPRE reporter, relative to the mutant Nluc 3xPREmt, was measured in HCT116 cells, in comparison to cells transfected with non-targeting control siRNAs. n=9; 3 experiments, each with 3 biological replicates; +/- SD.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed 3 times with 250 μl 1xPBS + 0.1% Tween-20 and incubated in 100 μl of 1xPBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Activity Assay, Luciferase, Binding Assay, Generated, Construct, Control, Transfection, Standard Deviation, Western Blot, Knockdown, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to RNA-binding protein-directed decay

doi: 10.1101/2025.10.02.680050

Figure Lengend Snippet: A. PABPC1 and PABPC4 co-immunoprecipitate with PUM1 from HCT116 cell extracts treated with RNases A and One. PUM1, eIF4E, eIF4G, PABPC1, PABPC4, and GAPDH were detected by western blot. B. Denaturing formaldehyde agarose gel analysis confirmed depletion of RNA in the HCT116 cell extracts before (-) or after treatment with RNase A and RNase One (+). The 18S and 28S ribosomal RNA bands, detected by ethidium bromide, are indicated on the right. C. Human PABPC1 domain architecture showing the N-terminal RRM domains (green) with the critical eIF4G binding site residues and the proline rich linker and C-terminal region PABC domain (blue) containing the MLLE motif residues important binding by PAM2-domain containing proteins. D. Western blot of the co-immunoprecipitation analysis of endogenous PUM1 with either HaloTag (HT), as a negative control, or HT-PABPC1 full-length (aa1-636) or HT-RRMs 1-4 (aa 1-370). All input samples were treated with RNase A and One. IgG beads and GAPDH served as negative controls. Dashed vertical lines in the panels indicate that the images were cropped to show relevant lanes. E. Western blot of PUM1 immunoprecipitates to detect association with either wild type full-length HT-PABPC1, or mutant versions wherein the eIF4G binding site is mutated (M161A and D165A) or the MLLE motif is mutated to (MLLEmt: M584G, L585A, L586A, and E587R), or the HT-MLLE domain (aa 542-636). All input samples were treated with RNase A and One.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed 3 times with 250 μl 1xPBS + 0.1% Tween-20 and incubated in 100 μl of 1xPBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Western Blot, Agarose Gel Electrophoresis, Binding Assay, Immunoprecipitation, Negative Control, Mutagenesis

Journal: bioRxiv

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to RNA-binding protein-directed decay

doi: 10.1101/2025.10.02.680050

Figure Lengend Snippet: A. Western blot analysis confirms auxin induced depletion of degron-tagged PABPC1-AID and RNAi depletion of PABPC4 either individually or in combination in HCT116 cells. RNAi was performed by transfecting cells with either PABPC4 or non-targeting control NTC siRNAs. PABPC1-AID protein was depleted upon treatment with indole-3-acetic acid (+IAA), in comparison to vehicle only control (-IAA). GAPDH served as a loading control. B. PUM-mediated repression of Nluc 3xPRE reporter expression was measured in HCT116 cells, relative to the mutant PRE version. Individual and combined effects of PABPC4 RNAi and IAA induced depletion of PABPC1-AID on PUM repression were tested. n=9; 3 experiments, each with 3 biological replicates; +/- SD. For significance calling, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = **** based on ordinary one-way ANOVA and Tukey test for multiple comparisons. C. Western blot analysis of samples from a representative experimental replicate for experiments shown in Panels D and E. GAPDH served as a loading control and PUM1 and PUM2 protein levels were detected as controls. The effect of combined PABPC4 RNAi and PABPC1-AID depletion on PUM repression of Nluc reporters containing the 3′ UTRs of the natural, PRE-containing, PUM target mRNAs FZD8 in panel D and CDKN1B in panel E were measured, relative to matched PRE mutant versions. n=9; 3 experiments, each with 3 biological replicates; +/- SD. Live cell numbers (panel F ) and viability (panel G ) were measured in control or PABPC1-AID and PABPC4 RNAi depletion conditions from reporter assays shown in panel B. n=12; 3 experiments, each with 2 biological replicates, 2 technical replicates; +/- SD.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed 3 times with 250 μl 1xPBS + 0.1% Tween-20 and incubated in 100 μl of 1xPBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Western Blot, Control, Comparison, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to RNA-binding protein-directed decay

doi: 10.1101/2025.10.02.680050

Figure Lengend Snippet: A. Expression levels of the PRE-containing PUM target mRNAs ITGA2 and SMPDL3A were measured by RTqPCR in RNA samples purified from HCT116 cells wherein PUM1 and PUM2 were depleted by RNAi. Changes in mRNA levels were determined relative to cells treated with the non-targeting control siRNAs (NTC). The non-targeted GAPDH mRNA and the non-adenylated MALAT1 non-coding RNA served as controls. B. Expression levels of the PRE-containing PUM target mRNAs ITGA2 and SMPDL3A were measured by RTqPCR in RNA samples purified from HCT116 cells wherein PABPC1-AID and PABPC4 were depleted by auxin treatment (+IAA) and RNAi, respectively. Changes in mRNA levels were determined relative to cells treated with vehicle only (-IAA) and the NTC siRNA. The non-targeted GAPDH mRNA and the non-adenylated MALAT1 non-coding RNA served as controls. In both panels A and B, transcript level was measured by RTqPCR, normalized to 18S rRNA, and plotted as fold change relative to the non-depleted control condition. n=3 biological replicates, ± SD. For significance calling, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = ****, ns = not significant based on unpaired two-tailed t-tests. C. Western blot analysis confirmed RNAi depletion of PUM1 and PUM2 in biological replicates in panel A. GAPDH served as a loading control. D. Western blot analysis of PABPC1-AID and PABPC4 confirmed their depletion by auxin induced degradation and RNAi, respectively, in samples from panel B. Histone H3 served as a loading control.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed 3 times with 250 μl 1xPBS + 0.1% Tween-20 and incubated in 100 μl of 1xPBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Expressing, Purification, Control, Two Tailed Test, Western Blot

Journal: bioRxiv

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to RNA-binding protein-directed decay

doi: 10.1101/2025.10.02.680050

Figure Lengend Snippet: A. Western blot analysis of halotag (HT) and HT-PABPC1 titration for samples from a representative experimental replicate of panel B. The amount of transfected plasmid for each effector is shown at the top. GAPDH served as a loading control. PUM1 and PUM2 were detected as an additional control. B. Reporter assay showing the effect of HT-PABPC1 over-expression on PUM repression of the Nluc 3xPRE reporter in wild type HCT116 cells, calculated relative to the Nluc 3xPREmt reporter. Halotag served as a negative control. n=9; 3 experiments, each with 3 biological replicates; +/- SD. For significance calling, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = **** based on ordinary one-way ANOVA and Tukey test for multiple comparisons. Significance indicated above the X-axis indicates relative to 3xPREmt reporter, whereas significance calling shown below is indicated by the respective brackets. C. Graph of the fold change in HT-PABPC1 exogenous expression over endogenous PABPC1 levels calculated from three experimental replicates including the western blot shown in panel A. n=3; +/- SD. D. Western blot analysis of halotag (HT), HT-PABPC1 full-length, HT-PABPC1 full-length RNA- binding mutant (RBmt), HT-RRM1-4 domains, and HT-RRM1-4 RBmt samples taken from a representative experimental replicate of panel E. GAPDH served as a loading control. PUM1 and PUM2 were detected as controls. E. Reporter assay showing effect of HT-PABPC1 full-length, HT-RRM1-4 domains, and RNA- binding mutants versions when over-expressed on PUM repression of the Nluc 3xPRE reporter in HCT116 cells. n=9; 3 experiments, each with 3 biological replicates; +/- SD. Halotag served as a negative control. F. Western blot analysis of over-expressed HT-PABPC1 on PUM repression and the effect of the mutation of the eIF4G binding site mutant (M161A, D165A), or the MLLE motif (MLLEmt), or the RNA-binding defective mutant (RBmt) from a representative experimental replicate of panel G. H3 served as a loading control. PUM1 and PUM2 were detected as additional controls. G. Reporter assay showing effect of HT-PABPC1 full-length mutants on PUM repression in wild type HCT116 cells. n=9; 3 experiments, each with 3 biological replicates; +/- SD. Halotag served as a negative control.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed 3 times with 250 μl 1xPBS + 0.1% Tween-20 and incubated in 100 μl of 1xPBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Western Blot, Titration, Transfection, Plasmid Preparation, Control, Reporter Assay, Over Expression, Negative Control, Expressing, RNA Binding Assay, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to RNA-binding protein-directed decay

doi: 10.1101/2025.10.02.680050

Figure Lengend Snippet: A. Co-immunoprecipitation analysis of PUM1 binding to the Nluc 3xPRE reporter in the presence of over-expressed halotag (HT) or HT-PABPC1. The top two panels show western blot detection of PUM1 protein and HT-PABPC1 in the 2% of the input HCT116 cell extracts the and 25% of the PUM1 RNA co-immunoprecipitates (RIP) from three biological replicate samples per condition. The bottom panel shows detection of Nluc mRNA by northern blotting. For northern blots, 1% of input samples and 75% of the RIP samples were loaded on the formaldehyde-MOPS agarose gel, respectively. Co-immunoprecipitation with IgG beads served as a negative control. B. Fold increase of the Nluc 3xPRE mRNA in the input samples from cells expressing HT- PABPC1 are plotted relative to the HT control, based on data in panel A. n=3 biological replicates, +/- SD. For significance calling, p < 0.001 = *** based on an unpaired student’s t-test. C. Fold enrichment of the Nluc 3xPRE mRNA in PUM1 RIP samples was measured relative to the IgG control. Importantly, the mRNA levels in each RIP sample was normalized to that present in the respective input samples. n=3 biological replicates, +/- SD. For significance calling, the difference between HT and HT-PABPC1 was not significant (p > 0.05 = ns) based on an unpaired student’s t-test.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed 3 times with 250 μl 1xPBS + 0.1% Tween-20 and incubated in 100 μl of 1xPBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Immunoprecipitation, Binding Assay, Western Blot, Northern Blot, Agarose Gel Electrophoresis, Negative Control, Expressing, Control