Journal: Journal of cell science

Article Title: IP6K1 upregulates the formation of processing bodies by influencing protein-protein interactions on the mRNA cap.

doi: 10.1242/jcs.259117

Figure Lengend Snippet: Fig. 4. IP6K1 interacts with the mRNA decapping complex on ribosomes. (A-C) Representative immunoblots examining the co-immunoprecipitation of endogenous DCP1A, PAN3, EDC4, DCP2 and DDX6 with SFB-tagged IP6K1. SFB-IP6K1 or SFB-GFP were transiently overexpressed in HEK293T cells, pulled down with either anti-FLAG antibody (A and C) or streptavidin sepharose beads (B), and probed to detect DCP1A, PAN3, EDC4, DCP2 or DDX6. The SFB tag was detected using an anti-FLAG antibody (N=3). There was some weak interaction of EDC4 and DCP2 with SFB-GFP, albeit lower than the binding of these proteins to SFB-IP6K1. (D) Representative immunoblots examining co-immunoprecipitation of endogenous DCP1A and DDX6 with endogenous IP6K1. A HEK293T cell extract was subjected to immunoprecipitation with an antibody directed against the N-terminal region of IP6K1, and probed to detect DCP1A or DDX6 (N=3). (E) Representative immunoblots of subcellular fractions of U-2 OS cells showing the detection of endogenous IP6K1, EDC4, DCP1A, and DCP2. Enrichment of ribosomes was marked by the presence of RPL6 and RPS6, and GAPDH was detected to rule out cytoplasmic contamination in the ribosomal fraction (N=4). The vertical line indicates the removal of non-essential lanes from a single original gel to improve visualization. (F,G) Representative immunoblots examining the co-immunoprecipitation of endogenous RPS6, DCP1A, PAN3, EDC4, DDX6 and DCP2 with SFB-tagged IP6K1 in ribosomes. The ribosomal fraction isolated from HEK293T cells transiently overexpressing SFB-IP6K1 or SFB-GFP was subjected to pull down with streptavidin sepharose beads, and then probed to detect the indicated proteins (N=3 for RPS6 and EDC4, and N=4 for DCP1A, DCP2 and DDX6). There was some weak interaction of DCP2 with SFB-GFP, albeit lower than its binding to SFB-IP6K1. The asterisks in G indicate specific bands.

Article Snippet: Plasmids expressing myc-tagged mouse IP6K1 (GenBank ID NM_013785.2), human IP6K2 (GenBank ID NM_001005909.3) and human IP6K3 (GenBank ID NM_001142883.2) were gifts from Solomon Snyder (Johns Hopkins School of Medicine, Baltimore, MD, USA). pT7-EGFP-C1-HsRCK (DDX6) (Addgene plasmid 25033; GenBank ID NM_004397), pCIneo-lambdaN-HA-HsEDC4 (Addgene plasmid 66597; GenBank ID NM_014329.4) and pT7-EGFPC1-HsDCP2 (Addgene plasmid 25031; GenBank ID AY146650) were gifts from Elisa Izaurralde (Max Planck Institute for Developmental Biology, Tübingen, Germany).

Techniques: Western Blot, Immunoprecipitation, Binding Assay, Isolation

Journal: Journal of cell science

Article Title: IP6K1 upregulates the formation of processing bodies by influencing protein-protein interactions on the mRNA cap.

doi: 10.1242/jcs.259117

Figure Lengend Snippet: Fig. 7. IP6K1 facilitates proteome remodelling on the mRNA cap to promote translational suppression. IP6K1 associates with the mRNA decapping complex via DCP2 and DDX6, and with the translation initiation complex via eIF4E. Other proteins that constitute the mRNA decapping complex and the translation initiation complex (shown inside light blue capsules) interact either directly or indirectly with IP6K1. The ability of IP6K1 to exhibit multi-protein interactions allows it to promote the assembly of proteins that facilitate translational suppression. Specifically, IP6K1 upregulates interaction between members of the translation suppression complex 4-ET and DDX6, and enhances binding of this complex with the cap binding protein eIF4E. IP6K1 also increases the binding of the decapping enhancer EDC4 to DDX6. These enhanced interactions subsequently lead to P-body formation. The protein TTP is also known as ZFP36. ARE, AU-rich element.

Article Snippet: Plasmids expressing myc-tagged mouse IP6K1 (GenBank ID NM_013785.2), human IP6K2 (GenBank ID NM_001005909.3) and human IP6K3 (GenBank ID NM_001142883.2) were gifts from Solomon Snyder (Johns Hopkins School of Medicine, Baltimore, MD, USA). pT7-EGFP-C1-HsRCK (DDX6) (Addgene plasmid 25033; GenBank ID NM_004397), pCIneo-lambdaN-HA-HsEDC4 (Addgene plasmid 66597; GenBank ID NM_014329.4) and pT7-EGFPC1-HsDCP2 (Addgene plasmid 25031; GenBank ID AY146650) were gifts from Elisa Izaurralde (Max Planck Institute for Developmental Biology, Tübingen, Germany).

Techniques: Capsules, Binding Assay

Journal: Microbiology Spectrum

Article Title: Long Noncoding RNA AROD Inhibits Host Antiviral Innate Immunity via the miR-324-5p–CUEDC2 Axis

doi: 10.1128/spectrum.04206-22

Figure Lengend Snippet: lnc-AROD targets Hsa-miR-324-5p. (A) RT-qPCR analysis of lnc-AROD in the nuclear and cytoplasmic fractions of A549 cells. MALAT1, GAPDH, U6, and RPS18 mRNA expression levels were used as controls. (B) RT-qPCR analysis of lnc-AROD expression in the nucleus and cytoplasm of H1N1 virus-infected A549 cells. (C) The cellular localization of lnc-AROD in A549 cells. The nuclei were stained with DAPI (blue), and the lnc-AROD probe was labeled with Cy3 (red). (D) AGO2 RIP was performed for detecting the amount of lnc-AROD in lnc-AROD-293T cells transfected with 3× Flag-AGO2 or 3× Flag-eGFP. IgG was used as the negative control. (E) RNAhybrid, starBase v2.0, TargetScan, and miRanda miRNA prediction programs consistently predicted four miRNAs interacted with lnc-AROD. (F) Luciferase activity of lnc-AROD in HEK293T cells transfected with miRNA mimics. (G and H) The plasmids pGLO-lnc-AROD and pGLO-lnc-AROD-del were generated to predict the miR-324-5p binding region. Luciferase activities of 293T cells transfected with hsa-miR-324-5p and pGLO-lnc-AROD or pGLO-lnc-AROD-del were quantified. (I and J) Bio-RNA pulldown and MS2-RIP assays were used to validate the interaction between hsa-miR-324-5p and lnc-AROD. (K) Colocalization between hsa-miR-324-5p and lnc-AROD was detected by RNA in situ hybridization in A549 cells. The data are expressed as means of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Argonaute2 (AGO2) was cloned into the PCDH-3flag-EGFP plasmid (catalog number 21538; Addgene), and lnc-AROD was cloned into the pMS2 plasmid, which was gifted from Myriam Gorospe ( ). lnc-AROD-specific siRNAs and CUEDC2-specific siRNAs were designed by Thermo RNAi Designer and transfected into human lung adenocarcinoma epithelial (A549) cells using jetPRIME transfection reagent (Polyplus, France) according to the manufacturer's instructions.

Techniques: Quantitative RT-PCR, Expressing, Virus, Infection, Staining, Labeling, Transfection, Negative Control, Luciferase, Activity Assay, Generated, Binding Assay, RNA In Situ Hybridization