Journal: The Journal of Biological Chemistry

Article Title: The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3′ UTR C-rich motifs

doi: 10.1016/j.jbc.2025.111042

Figure Lengend Snippet: hnRNP E1 silencing reduces p53 and p21 proteins but hnRNP E1 overexpression enhances p53 and p21 expression . A , bar graph showing the effect of siRNA-mediated silencing of hnRNP E1 on p53 and p21 mRNA levels in HepG2 cells, quantified by real-time PCR (RT-PCR). B , Western blot panels showing the effect of hnRNP E1 silencing on p53 and p21 protein levels in HepG2, MCF-7, A-549, and HEK-293 cells. Bar graph ( right ) shows normalized blot band intensities for hnRNP E1, p53, and p21. C , bar graph showing the effect of hnRNP E1 overexpression on p53 and p21 mRNA levels in HepG2 cells using RT-PCR. D , Western blot panels showing the effect of hnRNP E1 overexpression on the p53 and p21 protein levels in HepG2, MCF-7, A-549, and HEK-293 cells. Bar graph ( right ) represents normalized blot band intensities of hnRNP E1, p53, and p21. E , bar graph showing the effect of hnRNP E1 silencing on p53 and p21 mRNA levels in HuH7 cells using RT-PCR. F , Western blot panels showing the effect of hnRNP E1 silencing on p53 and p21 protein levels in Huh7 and MDA-MB-231 cells. Bar graph ( right ) shows normalized hnRNP E1, p53, and p21 band intensities. G , bar graph showing the effect of hnRNP E1 overexpression on p53 and p21 mRNA levels in Huh7 cells using RT-PCR. H , Western blot panels depicting the effect of hnRNP E1 overexpression on the p53 and p21 protein levels in Huh7 and MDA-MB-231 cells. Bar graph ( right ) shows the normalized band intensities of hnRNP E1, p53, and p21. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Experiments in A , C , E , and G were replicated thrice, and those in B , D , F , and H were repeated twice across cell types. hnRNP, heterogeneous nuclear ribonucleoprotein.

Article Snippet: For WT p53 overexpression, plasmid pcDNA3 p53 WT was used which was a gift from David Meek (Addgene plasmid # 69003; http://n2t.net/addgene : 69003; RRID: Addgene_69003).

Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3′ UTR C-rich motifs

doi: 10.1016/j.jbc.2025.111042

Figure Lengend Snippet: hnRNP E1 interacts with 3′-UTRs of p53 and p21 RNAs and enhances their stability . A – C , RNA–protein interaction heat maps generated using catRAPID, showing binding intensities of hnRNP E1 to the 3′-UTRs of p53 and p21; x -axis represents RNA nucleotide positions, and y -axis shows hnRNP E1 amino acid positions. Due to the large size of the p21 3′-UTR, the sequence was divided into proximal and distal segments for analysis. D , silver-stained SDS-PAGE gel showing the efficiency and specificity of RNA immunoprecipitation (RIP) using control (IgG) and V5 tag–based hnRNP E1 pulldown. E , agarose gel showing PCR-based confirmation of hnRNP E1 interactions with p53 and p21 RNAs in the RIP experiment. Dashed red boxes indicate the specific PCR-amplified products for p53 and p21. F and G , typhoon-scanned polyacrylamide gels from photoaffinity crosslinking experiment, showing direct interactions between 3′-UTRs of p53 and p21 with GST-hnRNP E1 fusion protein. Bands corresponding to the protein–RNA complexes are indicated with ∗. Lane 1 represents the blank control. No interaction was observed between the 5′-UTRs of either p53 or p21 with purified GST-hnRNP E1 fusion protein. H and I , mRNA decay assay for p53 and p21 under hnRNP E1-silenced conditions in HepG2 cells using actinomycin D, showing reduced mRNA stability upon hnRNP E1 depletion. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Data ( F – I ) originated from three independent experiments. hnRNP, heterogeneous nuclear ribonucleoprotein.

Article Snippet: For WT p53 overexpression, plasmid pcDNA3 p53 WT was used which was a gift from David Meek (Addgene plasmid # 69003; http://n2t.net/addgene : 69003; RRID: Addgene_69003).

Techniques: Generated, Binding Assay, Sequencing, Staining, SDS Page, RNA Immunoprecipitation, Control, Agarose Gel Electrophoresis, Amplification, Purification, Mrna Decay Assay

Journal: The Journal of Biological Chemistry

Article Title: The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3′ UTR C-rich motifs

doi: 10.1016/j.jbc.2025.111042

Figure Lengend Snippet: hnRNP E1 binds to C-rich RNA motifs on 3′-UTRs of p53 and p21 and modulates their regulation . A and B , predicted hnRNP E1 binding sites on p53 and p21 3′-UTRs were identified using catRAPID and are illustrated in schematic models of the respective mRNAs. C and D , typhoon-scanned polyacrylamide gels from photoaffinity crosslinking experiments show interactions between GST-hnRNP E1 fusion protein and WT 3′-UTRs of both p53 and p21. Bands corresponding to the interacting protein–RNA complexes are indicated with ∗. Motif-deleted mutant 3′-UTRs [3′-UTR(CY5) Mut; p53 Δ-motif 1/2/3/4 and p21 Δ-motif 1/2/3] showed no interaction (last lanes, both the gels). Lane 1 represents a blank control. No interaction of GST was observed with the 3′-UTRs of either p53 or p21. E and F , bar graph showing the functional role of each identified motif in the context of 3′-UTR–guided functional regulation of p53 and p21, by RT-PCR–based luciferase RNA levels and luciferase assays. HepG2 cell were transfected with either p53 or p21 5′-UTR-luciferase-3′-UTR expression plasmids or motif-deleted variants under hnRNP E1-silenced conditions (sihnRNP E1-mediated knockdown). Luciferase RNA levels and luciferase activity from siControl-transfected cells was normalized to 1, and relative luciferase RNA level and activity of WT and MUT (motif-deleted variants) 3′-UTRs is shown. The ratio of the normalized luciferase values to the normalized luciferase RNA levels are shown in the boxes below the bar graph. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. Data represents findings from three independent experimental replicates. hnRNP, heterogeneous nuclear ribonucleoprotein.

Article Snippet: For WT p53 overexpression, plasmid pcDNA3 p53 WT was used which was a gift from David Meek (Addgene plasmid # 69003; http://n2t.net/addgene : 69003; RRID: Addgene_69003).

Techniques: Binding Assay, Mutagenesis, Control, Functional Assay, Reverse Transcription Polymerase Chain Reaction, Luciferase, Transfection, Expressing, Knockdown, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3′ UTR C-rich motifs

doi: 10.1016/j.jbc.2025.111042

Figure Lengend Snippet: hnRNP E1 induces translation of p53 and p21 mRNA via their 3′-UTRs . A and B , bar graph showing the normalized (by setting control to 1) luciferase RNA levels using RT-PCR and luciferase assay–based translational efficiency under hnRNP E1-silenced or hnRNP E1-overexpressed cellular conditions in HepG2 cells transfected with p53 5′-UTR-luciferase-3′-UTR expression plasmid. C and D , bar graph showing the normalized (by setting control to 1) luciferase RNA levels using RT-PCR and luciferase assay–based translational efficiency under hnRNP E1-silenced or hnRNP E1-overexpressed cellular conditions in HepG2 cells transfected with p21 5′-UTR-luciferase-3′-UTR expression plasmid. Values in the boxes below the bar graph (A to D) indicate the ratio of normalized luciferase activity to luciferase RNA. E and F , line graph showing the in vitro translation assays of in vitro runoff transcripts containing both the 5′- and 3′-UTRs of p53 and p21 in the presence of increasing concentrations of purified GST-hnRNP E1 (GST-E1), showing time-dependent luciferase activity. G and H , similar to the experiment E and F, the in vitro translation assay for in vitro runoff transcripts containing only 5′-UTRs of p53 and p21. I and J , similar to the experiment E and F, the in vitro translation assay for in vitro runoff transcripts containing only 3′-UTRs of p53 and p21. All the experimental time points (5, 15, 30, 60, and 90 min) are presented in a linear scale. Schematic representations of transcript configurations (E to J) are provided. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Experiments were performed in triplicate. hnRNP, heterogeneous nuclear ribonucleoprotein.

Article Snippet: For WT p53 overexpression, plasmid pcDNA3 p53 WT was used which was a gift from David Meek (Addgene plasmid # 69003; http://n2t.net/addgene : 69003; RRID: Addgene_69003).

Techniques: Control, Luciferase, Reverse Transcription Polymerase Chain Reaction, Transfection, Expressing, Plasmid Preparation, Activity Assay, In Vitro, Purification

Journal: The Journal of Biological Chemistry

Article Title: The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3′ UTR C-rich motifs

doi: 10.1016/j.jbc.2025.111042

Figure Lengend Snippet: hnRNP E1 induces p53 and p21 mRNA polyribosome loading . A and B , ribosomal profiles from sucrose density gradient fractionation from HepG2 cells overexpressing or silenced for hnRNP E1. Fractionation efficiency demonstrated by probing for RPS6 and RPL11 distributions across alternative fractions with western blots. The hnRNP E1 silencing and overexpression are shown in the Western blot panels ( top right of each subfigure). C and D , semiquantitative midphase PCR showing the relative abundance of p53, p21, and β-actin (control) mRNAs, in the collected alternative fractions from polyribosome fractionation assay following RNA extraction and cDNA synthesis. E and F , line graphs showing the relative band intensities of the p53, p21, and β-actin cDNA amplicons under hnRNP E1–overexpressed and hnRNP E1-silenced conditions relative to the control (normalized to 1). Data originated from three independent experiments. hnRNP, heterogeneous nuclear ribonucleoprotein. cDNA, complementary DNA.

Article Snippet: For WT p53 overexpression, plasmid pcDNA3 p53 WT was used which was a gift from David Meek (Addgene plasmid # 69003; http://n2t.net/addgene : 69003; RRID: Addgene_69003).

Techniques: Fractionation, Western Blot, Over Expression, Control, RNA Extraction, cDNA Synthesis

Journal: The Journal of Biological Chemistry

Article Title: The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3′ UTR C-rich motifs

doi: 10.1016/j.jbc.2025.111042

Figure Lengend Snippet: KH1 and KH2 domains of hnRNP E1 mediates p53/p21 expression . A , secondary structures of the KH domains of hnRNP E1 is shown. Dot matrix alignment of the three consensus KH domains (accession numbers NP_006187.2 ; CAA55016.1 ; NM_006196 ; BC039742 and X78137 ) is shown, where matches are shown as dots and the deletions/gaps as dashes . For positions of three α-helices ( blue ) and three β-sheets ( red ) of each KH domain are indicated with dashed boxes on the aligned sequences. B , sequence identity matrix showing the degree of sequence similarity among the three consensus KH domains of hnRNP E1. Matrix was generated using BioEdit software (version 7.2; https://bioedit.software.informer.com/7.2/#google_vignette ) after excluding the indel sites. C , pictorial map depicting the location of three KH domains of hnRNP E1 and different KH-deleted variants of the hnRNP E1 expression system with V5 and SBP tags. D , intracellular localization of full-length hnRNP E1 and KH domain–deleted variants detected by immunofluorescence assay. The scale bar represents 10 μm. E , bar graph showing the relative expression of p53 and p21 RNA in cellular conditions overexpressing full-length hnRNP E1 or its different KH-deleted variants, measured by realtime PCR assay. F , Western blot analysis of p53 and p21 protein levels under overexpression of full-length hnRNP E1 or its different KH-deleted variants. Bar graph ( right ) shows normalized protein band intensities of p53 and p21. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. The experiments ( D to F ) were replicated thrice. hnRNP, heterogeneous nuclear ribonucleoprotein.

Article Snippet: For WT p53 overexpression, plasmid pcDNA3 p53 WT was used which was a gift from David Meek (Addgene plasmid # 69003; http://n2t.net/addgene : 69003; RRID: Addgene_69003).

Techniques: Expressing, Sequencing, Generated, Software, Immunofluorescence, Western Blot, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3′ UTR C-rich motifs

doi: 10.1016/j.jbc.2025.111042

Figure Lengend Snippet: KH1 and KH2 domains of hnRNP E1 independently binds to 3′-UTRs of p53/p21 and modulate their translation . A , different KH domains with V5 and SBP tags are shown in the figure. B , Western blots showing nuclear versus cytoplasmic distribution of full-length hnRNP E1 and its KH domains based on fractionation assays. C , intracellular localization of hnRNP E1 and its KH domains detected by immunofluorescence assay. The scale bar represents 10 μm. Bar graph ( bottom ) shows the relative cytoplasmic localization scores normalized to nuclear localization score (set to 1). D , bar graph showing the relative expression of p53 and p21 RNA in cellular conditions overexpressing full-length hnRNP E1 or its different KH domains measured by real-time PCR. E , Western blot analysis of p53 and p21 protein levels under overexpression of full-length hnRNP E1 or its KH domains. Bar graph ( right ) displays the corresponding normalized protein band intensities. F and G , bar graph showing the effect of different KH domains on the translational efficiency measured using luciferase assays. HepG2 cells were cotransfected with each KH domain–encoding plasmids and either p53 5′-UTR-luciferase-3′-UTR ( F ) or with p21 5′-UTR-luciferase-3′-UTR ( G ) expression plasmids. H and I , typhoon-scanned polyacrylamide gels of the photoaffinity crosslinking experiments showing interactions of 3′-UTRs of p53 and p21 with GST-KH fusion proteins. Bands corresponding to the proteins–RNA complexes are indicated with ∗. Lane 1, blank control; GST-hnRNP E1 (full-length), served as positive control. J and K , line graph showing the time-dependent in vitro translation efficiency (luciferase signal) of in vitro runoff transcripts containing both 5′- and 3′-UTRs of p53 and p21, respectively, in the presence of purified GST-hnRNP E1 (GST-E1) and GST-KH (1/2/3) fusion proteins at equal concentrations (100 nM). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. The experiments ( C – K ) were performed in triplicate. hnRNP, heterogeneous nuclear ribonucleoprotein.

Article Snippet: For WT p53 overexpression, plasmid pcDNA3 p53 WT was used which was a gift from David Meek (Addgene plasmid # 69003; http://n2t.net/addgene : 69003; RRID: Addgene_69003).

Techniques: Western Blot, Fractionation, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction, Over Expression, Luciferase, Control, Positive Control, In Vitro, Purification