m7 gpppa rna cap structure analog  (New England Biolabs)


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    Structured Review

    New England Biolabs m7 gpppa rna cap structure analog
    Structural and mutagenesis analysis of RNase AM activities. (A) Overall crystal <t>structure</t> of RNase AM PHP domain (teal) with manganese ions (purple) and sulfate (yellow and red) shown as spheres. (B) Overlay of RNase AM crystal structure (teal) with RNase AM from AlphaFold (light blue) showing the insertion domain. (C) Overlay of RNase AM with CV1693 (gray) with AMP from CV1693 shown as sticks. (D) The active site of RNase AM showing the coordination of the three manganese ions and sulfate to the active site residues (sticks). AMP and manganese (gray) and the phosphate (orange and red) from the overlay with CV1693 are shown. (E) Electrostatic potential mapped onto the surface of RNase AM showing the positively charged region where the 5’P from CV1693 AMP (gray) is buried. (F) Comparison of binding of AMP (gray) between RNase AM and CV1693 showing conservation of the residues that interact with AMP. (G) Reaction products of in vitro deFADding assays with 100 nM recombinant RNase AM and putative catalytically inactive point mutants. Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped <t>RNA</t> (FAD–) are indicated. The asterisk represents the 32 P-labeling within the body of the RNA. Reaction products were resolved on 15% 7M urea PAGE gels
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    Images

    1) Product Images from "Identification of a novel deFADding activity in 5’ to 3’ exoribonucleases"

    Article Title: Identification of a novel deFADding activity in 5’ to 3’ exoribonucleases

    Journal: bioRxiv

    doi: 10.1101/2022.05.10.491372

    Structural and mutagenesis analysis of RNase AM activities. (A) Overall crystal structure of RNase AM PHP domain (teal) with manganese ions (purple) and sulfate (yellow and red) shown as spheres. (B) Overlay of RNase AM crystal structure (teal) with RNase AM from AlphaFold (light blue) showing the insertion domain. (C) Overlay of RNase AM with CV1693 (gray) with AMP from CV1693 shown as sticks. (D) The active site of RNase AM showing the coordination of the three manganese ions and sulfate to the active site residues (sticks). AMP and manganese (gray) and the phosphate (orange and red) from the overlay with CV1693 are shown. (E) Electrostatic potential mapped onto the surface of RNase AM showing the positively charged region where the 5’P from CV1693 AMP (gray) is buried. (F) Comparison of binding of AMP (gray) between RNase AM and CV1693 showing conservation of the residues that interact with AMP. (G) Reaction products of in vitro deFADding assays with 100 nM recombinant RNase AM and putative catalytically inactive point mutants. Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped RNA (FAD–) are indicated. The asterisk represents the 32 P-labeling within the body of the RNA. Reaction products were resolved on 15% 7M urea PAGE gels
    Figure Legend Snippet: Structural and mutagenesis analysis of RNase AM activities. (A) Overall crystal structure of RNase AM PHP domain (teal) with manganese ions (purple) and sulfate (yellow and red) shown as spheres. (B) Overlay of RNase AM crystal structure (teal) with RNase AM from AlphaFold (light blue) showing the insertion domain. (C) Overlay of RNase AM with CV1693 (gray) with AMP from CV1693 shown as sticks. (D) The active site of RNase AM showing the coordination of the three manganese ions and sulfate to the active site residues (sticks). AMP and manganese (gray) and the phosphate (orange and red) from the overlay with CV1693 are shown. (E) Electrostatic potential mapped onto the surface of RNase AM showing the positively charged region where the 5’P from CV1693 AMP (gray) is buried. (F) Comparison of binding of AMP (gray) between RNase AM and CV1693 showing conservation of the residues that interact with AMP. (G) Reaction products of in vitro deFADding assays with 100 nM recombinant RNase AM and putative catalytically inactive point mutants. Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped RNA (FAD–) are indicated. The asterisk represents the 32 P-labeling within the body of the RNA. Reaction products were resolved on 15% 7M urea PAGE gels

    Techniques Used: Mutagenesis, Binding Assay, In Vitro, Recombinant, Labeling, Polyacrylamide Gel Electrophoresis

    Xrn1 deFADs FAD-capped RNAs in both yeast and human cells. (A) Time-course decay analysis of uniformly 32 P-labeled m 7 G- or FAD-capped RNA in the presence of cell extract prepared from WT or xrn1Δ strains. The remaining RNA was quantified and plotted from three independent experiments with ± SD denoted by error bars. (B) FAD-capQ assay using different amounts of in vitro transcribed FAD-capped RNA to demonstrate the release of intact FAD upon Xrn1 mediated deFADding. Catalytic-dead mutant of Xrn1, E178Q was used as a control for the Xrn1 reaction and NAD-capped RNA was used as a control for specific detection of FAD in the assay (C) Total RNAs from WT and xrn1Δ were subjected to the FAD-capQ assay to detect total levels of cellular FAD-capped RNA. Data represents average from three independent experiments. Error bars represent ± SD with unpaired t -test; * p
    Figure Legend Snippet: Xrn1 deFADs FAD-capped RNAs in both yeast and human cells. (A) Time-course decay analysis of uniformly 32 P-labeled m 7 G- or FAD-capped RNA in the presence of cell extract prepared from WT or xrn1Δ strains. The remaining RNA was quantified and plotted from three independent experiments with ± SD denoted by error bars. (B) FAD-capQ assay using different amounts of in vitro transcribed FAD-capped RNA to demonstrate the release of intact FAD upon Xrn1 mediated deFADding. Catalytic-dead mutant of Xrn1, E178Q was used as a control for the Xrn1 reaction and NAD-capped RNA was used as a control for specific detection of FAD in the assay (C) Total RNAs from WT and xrn1Δ were subjected to the FAD-capQ assay to detect total levels of cellular FAD-capped RNA. Data represents average from three independent experiments. Error bars represent ± SD with unpaired t -test; * p

    Techniques Used: Labeling, In Vitro, Mutagenesis

    RNase AM is a novel deFADding enzyme in E. coli. (A) Reaction products of in vitro deFADding assays with 100nM recombinant RNase AM. Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped RNA (FAD–), NAD-capped (NAD–), and m 7 G-capped (m 7 G–). The asterisk represents the 32 P-labeling within the body of the RNA. Reaction products were resolved on 15% 7M urea PAGE gels. (B) Time-course decay analysis of uniformly 32 P-labeled monophosphate or FAD-capped RNA with the indicated amount of RNase AM protein are shown. Quantitation of RNA remaining is plotted from three independent experiments with ± SD denoted by error bars. (C) To demonstrate that RNase AM releases intact FAD upon deFADding like Xrn1 (in Fig 3 ), FAD-capQ assay using different amount of in vitro transcribed FAD-capped RNA was used. Catalytic-dead mutant of RNase AM, D20A was used as control for the reaction. (D) Total RNA from WT and rnase AMΔ were subjected to the FAD-capQ assay to detect total levels of cellular FAD-capped RNA. Data represents an average from three independent experiments. Error bars represent ± SD with unpaired t -test; * p
    Figure Legend Snippet: RNase AM is a novel deFADding enzyme in E. coli. (A) Reaction products of in vitro deFADding assays with 100nM recombinant RNase AM. Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped RNA (FAD–), NAD-capped (NAD–), and m 7 G-capped (m 7 G–). The asterisk represents the 32 P-labeling within the body of the RNA. Reaction products were resolved on 15% 7M urea PAGE gels. (B) Time-course decay analysis of uniformly 32 P-labeled monophosphate or FAD-capped RNA with the indicated amount of RNase AM protein are shown. Quantitation of RNA remaining is plotted from three independent experiments with ± SD denoted by error bars. (C) To demonstrate that RNase AM releases intact FAD upon deFADding like Xrn1 (in Fig 3 ), FAD-capQ assay using different amount of in vitro transcribed FAD-capped RNA was used. Catalytic-dead mutant of RNase AM, D20A was used as control for the reaction. (D) Total RNA from WT and rnase AMΔ were subjected to the FAD-capQ assay to detect total levels of cellular FAD-capped RNA. Data represents an average from three independent experiments. Error bars represent ± SD with unpaired t -test; * p

    Techniques Used: In Vitro, Recombinant, Labeling, Polyacrylamide Gel Electrophoresis, Quantitation Assay, Mutagenesis

    Identification of FAD cap binding proteins in budding yeast. (A) Schematic illustration of the FAD cap -RNA Affinity Purification (FcRAP). (B) Eluates from FcRAP were loaded on to a 4%-12% Bis-Tris gel and stained with SYPRO Ruby. The m 7 G cap affinity purification was used as a control. All protein bands labeled on the gel were excised from the gel and identified using mass spectrometry. (C) Electrophoretic mobility shift assay (EMSA) using increasing concentration of Xrn1 (2.5 pmol, 5 pmol and 7.5 pmol) and a fixed concentration (5 pmol) of uniformly labeled in vitro transcribed FADcapped or NAD-capped or triphosphate RNA (pppA–). (D) EMSA using equimolar concentration (5pmol) of Xrn1 and uniformly labeled in vitro transcribed FAD-capped or NAD-capped or pppA–RNAs in the presence of total yeast RNA as a nonspecific competitor.
    Figure Legend Snippet: Identification of FAD cap binding proteins in budding yeast. (A) Schematic illustration of the FAD cap -RNA Affinity Purification (FcRAP). (B) Eluates from FcRAP were loaded on to a 4%-12% Bis-Tris gel and stained with SYPRO Ruby. The m 7 G cap affinity purification was used as a control. All protein bands labeled on the gel were excised from the gel and identified using mass spectrometry. (C) Electrophoretic mobility shift assay (EMSA) using increasing concentration of Xrn1 (2.5 pmol, 5 pmol and 7.5 pmol) and a fixed concentration (5 pmol) of uniformly labeled in vitro transcribed FADcapped or NAD-capped or triphosphate RNA (pppA–). (D) EMSA using equimolar concentration (5pmol) of Xrn1 and uniformly labeled in vitro transcribed FAD-capped or NAD-capped or pppA–RNAs in the presence of total yeast RNA as a nonspecific competitor.

    Techniques Used: Binding Assay, Affinity Purification, Staining, Labeling, Mass Spectrometry, Electrophoretic Mobility Shift Assay, Concentration Assay, In Vitro

    Xrn1 and Rat1 are deFADding enzymes. (A) Chemical structure of FAD-capped RNA. (B) Reaction products of in vitro deFADding assays with 50 nM recombinant Xrn1, WT, and catalytically inactive ( E178Q ) from K. lactis or 100 nM recombinant Rat1, WT or catalytically inactive ( E207Q ) from S. pombe or 25 nM Rai1, WT catalytically inactive ( D201A) (S. pombe ). Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped RNA (FAD–) where the line denotes the RNA were used as indicated. The asterisk represents the 32 P-labeling within the body of the RNA. Reaction products were resolved on 15% 7M urea PAGE gels. Time-course decay analysis of uniformly 32 P-labeled monophosphate or FAD-capped RNA with the indicated amount of Xrn1 (C) or Rat1 (D) protein are shown. Quantitation of RNA remaining is plotted from three independent experiments with ± SD denoted by error bars.
    Figure Legend Snippet: Xrn1 and Rat1 are deFADding enzymes. (A) Chemical structure of FAD-capped RNA. (B) Reaction products of in vitro deFADding assays with 50 nM recombinant Xrn1, WT, and catalytically inactive ( E178Q ) from K. lactis or 100 nM recombinant Rat1, WT or catalytically inactive ( E207Q ) from S. pombe or 25 nM Rai1, WT catalytically inactive ( D201A) (S. pombe ). Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped RNA (FAD–) where the line denotes the RNA were used as indicated. The asterisk represents the 32 P-labeling within the body of the RNA. Reaction products were resolved on 15% 7M urea PAGE gels. Time-course decay analysis of uniformly 32 P-labeled monophosphate or FAD-capped RNA with the indicated amount of Xrn1 (C) or Rat1 (D) protein are shown. Quantitation of RNA remaining is plotted from three independent experiments with ± SD denoted by error bars.

    Techniques Used: In Vitro, Recombinant, Labeling, Polyacrylamide Gel Electrophoresis, Quantitation Assay

    2) Product Images from "Identification of SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of the nsp14 RNA Cap Methyltransferase"

    Article Title: Identification of SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of the nsp14 RNA Cap Methyltransferase

    Journal: bioRxiv

    doi: 10.1101/2021.04.07.438810

    K m values for substrates of nsp14 a) Titration of various nsp14 susbstrates. GTP, GpppA cap analogue, GpppA-RNA, and me 7 GpppA cap analogue were incubated for 20 minutes with 5 nM nsp14 in the presence of 1 μM SAM. b-d) Determination of Michaelis constants for ( b ) GpppA-RNA, ( c ) GpppA, and ( d ) GTP for nsp14. nsp14 concentration was fixed at 5 nM for all experiments, and substrate concentration varied. V max values cannot be compared between substrates, and are therefore not given. HTRF values are given as raw, and not normalised, HTRF values. Errors given are 95% confidence ranges.
    Figure Legend Snippet: K m values for substrates of nsp14 a) Titration of various nsp14 susbstrates. GTP, GpppA cap analogue, GpppA-RNA, and me 7 GpppA cap analogue were incubated for 20 minutes with 5 nM nsp14 in the presence of 1 μM SAM. b-d) Determination of Michaelis constants for ( b ) GpppA-RNA, ( c ) GpppA, and ( d ) GTP for nsp14. nsp14 concentration was fixed at 5 nM for all experiments, and substrate concentration varied. V max values cannot be compared between substrates, and are therefore not given. HTRF values are given as raw, and not normalised, HTRF values. Errors given are 95% confidence ranges.

    Techniques Used: Titration, Incubation, Concentration Assay

    Nsp14 inhibitors do not inhibit nsp10-16 a) Gel filtration from final purification step of the nsp10-16 fusion protein. Coomassie shows fractions taken across the major peak of the gel filtration elution. Only fractions indicated were pooled (black bar, upper). Expected size of nsp10-16: 47.8 kDa (nsp10 - 13.3 kDa + nsp16 - kDa 34.5 kDa) b) Time course of the nsp10-16 methyltransferase reaction with 100 nM, 50 nM, 25 nM and 12.5 nM nsp10-16 enzyme. Reaction was conducted with 1.3 μM me 7 GpppA-RNA and 1 μM SAM. c) Determination of Michaelis constants for me 7 GpppA-RNA for nsp10-16. nsp10-16 concentration was fixed at 100 nM and substrate concentration varied. Errors given are 95% confidence ranges. d) Cross validation of nsp14 inhibitors with nsp10-16. Normalised HTRF values for nsp10-16 with inhibitors identified for nsp14 and sinefungin. All compounds tested at 50 μM. Reactions conducted with 1.3 μM me 7 GpppA-RNA and 1 μM SAM.
    Figure Legend Snippet: Nsp14 inhibitors do not inhibit nsp10-16 a) Gel filtration from final purification step of the nsp10-16 fusion protein. Coomassie shows fractions taken across the major peak of the gel filtration elution. Only fractions indicated were pooled (black bar, upper). Expected size of nsp10-16: 47.8 kDa (nsp10 - 13.3 kDa + nsp16 - kDa 34.5 kDa) b) Time course of the nsp10-16 methyltransferase reaction with 100 nM, 50 nM, 25 nM and 12.5 nM nsp10-16 enzyme. Reaction was conducted with 1.3 μM me 7 GpppA-RNA and 1 μM SAM. c) Determination of Michaelis constants for me 7 GpppA-RNA for nsp10-16. nsp10-16 concentration was fixed at 100 nM and substrate concentration varied. Errors given are 95% confidence ranges. d) Cross validation of nsp14 inhibitors with nsp10-16. Normalised HTRF values for nsp10-16 with inhibitors identified for nsp14 and sinefungin. All compounds tested at 50 μM. Reactions conducted with 1.3 μM me 7 GpppA-RNA and 1 μM SAM.

    Techniques Used: Filtration, Purification, Concentration Assay

    An HTRF based assay for methyltransferase activity a) Outline of the HTRF based assay for methyltransferase activity. Both nsp14 and nsp10-16 are SAM dependent methyltransferases that produce SAH following successful methyltransfer to their substrate. This SAH displaces SAH-d2 from the variable region of an α-SAH Tb cryptate-conjugated antibody, thus lowering HTRF signal through the disruption of the Tb cryptate – d2 FRET pair. b) nsp14 was assayed for methyltransferase activity through the HTRF based assay. The methyltransferase reaction was run in either the absence of 10nM nsp14, 1 μM SAM, 0.11 mM GpppA-RNA, or in the presence of all three components. In addition, the methyltransferase reaction was conducted in the presence of 0.25 μM, 2.5 μM and 25 μM of the pan-methyltransferase inhibitor Sinefungin, which acts as a competitive inhibitor (with respect to SAM) towards SAM-dependent methyltransferases. c) Time course of nsp14 activity by HTRF assay. 20 nM of nsp14 was incubated with 0.11 mM GpppA cap analogue and 1 μM SAM for the time indicated. In addition, an experiment was run in the absence of nsp14, and in the presence of the methyltransferase inhibitor sinefungin. The reaction was started as a master mix with the addition of nsp14, and 8 μl was removed at every time point and added to 2 μl of 5M NaCl to stop the methyltransferase reaction. Points are the mean of three technical repeats, and error bars indicate range.
    Figure Legend Snippet: An HTRF based assay for methyltransferase activity a) Outline of the HTRF based assay for methyltransferase activity. Both nsp14 and nsp10-16 are SAM dependent methyltransferases that produce SAH following successful methyltransfer to their substrate. This SAH displaces SAH-d2 from the variable region of an α-SAH Tb cryptate-conjugated antibody, thus lowering HTRF signal through the disruption of the Tb cryptate – d2 FRET pair. b) nsp14 was assayed for methyltransferase activity through the HTRF based assay. The methyltransferase reaction was run in either the absence of 10nM nsp14, 1 μM SAM, 0.11 mM GpppA-RNA, or in the presence of all three components. In addition, the methyltransferase reaction was conducted in the presence of 0.25 μM, 2.5 μM and 25 μM of the pan-methyltransferase inhibitor Sinefungin, which acts as a competitive inhibitor (with respect to SAM) towards SAM-dependent methyltransferases. c) Time course of nsp14 activity by HTRF assay. 20 nM of nsp14 was incubated with 0.11 mM GpppA cap analogue and 1 μM SAM for the time indicated. In addition, an experiment was run in the absence of nsp14, and in the presence of the methyltransferase inhibitor sinefungin. The reaction was started as a master mix with the addition of nsp14, and 8 μl was removed at every time point and added to 2 μl of 5M NaCl to stop the methyltransferase reaction. Points are the mean of three technical repeats, and error bars indicate range.

    Techniques Used: HTRF Assay, Activity Assay, Incubation

    3) Product Images from "The PBDE metabolite 6-OH-BDE 47 affects melanin pigmentation and THRβ MRNA expression in the eye of zebrafish embryos"

    Article Title: The PBDE metabolite 6-OH-BDE 47 affects melanin pigmentation and THRβ MRNA expression in the eye of zebrafish embryos

    Journal: Endocrine disruptors (Austin, Tex.)

    doi: 10.4161/23273739.2014.969072

    Effect of T3 on THRβ mRNA expression in the periventricular zone of zebrafish embryonic brain. Embryos were exposed from 4- to 22- hpf to T3. Note the prominent blue coloration in control, ( A ) localized to fore- and midbrain regions (red arrow). By comparison, panels B,C and E show less blue coloration (red arrows) upon exposure to a range of T3 from 0.1 nM to 100 nM. The average intensity of THRβ expression recorded in brain at 22 somite stage showed dose dependent reduction (histogram, E ). ( F ) Bar chart of THRβ expression based on quantitative PCR (qRT-PCR, total RNA from whole embryos) at 22 somite stage. The 10 and 100 nM T3 was significantly different from the control (*, P
    Figure Legend Snippet: Effect of T3 on THRβ mRNA expression in the periventricular zone of zebrafish embryonic brain. Embryos were exposed from 4- to 22- hpf to T3. Note the prominent blue coloration in control, ( A ) localized to fore- and midbrain regions (red arrow). By comparison, panels B,C and E show less blue coloration (red arrows) upon exposure to a range of T3 from 0.1 nM to 100 nM. The average intensity of THRβ expression recorded in brain at 22 somite stage showed dose dependent reduction (histogram, E ). ( F ) Bar chart of THRβ expression based on quantitative PCR (qRT-PCR, total RNA from whole embryos) at 22 somite stage. The 10 and 100 nM T3 was significantly different from the control (*, P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    4) Product Images from "Loquacious modulates flaviviral RNA replication in mosquito cells"

    Article Title: Loquacious modulates flaviviral RNA replication in mosquito cells

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1010163

    Effect of Loqs depletion on DENV RNA translation, replication and stability. (A) Western blot analysis of NS3, Loqs and GAPDH protein abundances in cytosolic and ER membrane fractions isolated from DENV2-infected Aag2 cell lysates at 72 hrs post infection. Representative image from three independent experiments is shown. (B) Immunoprecipitation of HA-tagged Loqs from Aag2 cells infected with DENV2 at a MOI of 1. Aag2 cells transfected with HA-GFP or HA-Loqs PA/PB plasmids were infected with DENV2 for 72 hrs and immunoprecipitations were performed with anti-HA antibody with or without RNaseA/T1 treatment. The abundances of DENV NS3, NS4B and capsid proteins in the immunoprecipitated material (IP) and the input lysates (10%) were determined by western blot analysis. (C) Luciferase activities of wildtype and replication-defective DENV2 luciferase replicons in C6/36 cells transfected with control siRNAs or siRNAs against Loqs (siLoqs-4 and siLoqs-5 were used at a final concentration of 25nM each). C6/36 cells were transfected with the indicated siRNAs followed by wildtype or replication-defective (NS5-GDD) replicon RNAs and harvested at the indicated time points. Average luciferase expression from DENV replicons from six independent replicates is shown (***p
    Figure Legend Snippet: Effect of Loqs depletion on DENV RNA translation, replication and stability. (A) Western blot analysis of NS3, Loqs and GAPDH protein abundances in cytosolic and ER membrane fractions isolated from DENV2-infected Aag2 cell lysates at 72 hrs post infection. Representative image from three independent experiments is shown. (B) Immunoprecipitation of HA-tagged Loqs from Aag2 cells infected with DENV2 at a MOI of 1. Aag2 cells transfected with HA-GFP or HA-Loqs PA/PB plasmids were infected with DENV2 for 72 hrs and immunoprecipitations were performed with anti-HA antibody with or without RNaseA/T1 treatment. The abundances of DENV NS3, NS4B and capsid proteins in the immunoprecipitated material (IP) and the input lysates (10%) were determined by western blot analysis. (C) Luciferase activities of wildtype and replication-defective DENV2 luciferase replicons in C6/36 cells transfected with control siRNAs or siRNAs against Loqs (siLoqs-4 and siLoqs-5 were used at a final concentration of 25nM each). C6/36 cells were transfected with the indicated siRNAs followed by wildtype or replication-defective (NS5-GDD) replicon RNAs and harvested at the indicated time points. Average luciferase expression from DENV replicons from six independent replicates is shown (***p

    Techniques Used: Western Blot, Isolation, Infection, Immunoprecipitation, Transfection, Luciferase, Concentration Assay, Expressing

    Diagram of the dengue viral (DENV) genome and strategy for the RNA-protein interaction (RAPID) assay. (A) DENV genome organization. The open reading frame encoding the three structural (C (capsid), prM/M (membrane), E (envelope)) proteins and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) proteins flanked by 5’ and 3’ untranslated regions (UTR) are shown. The 3’ UTR is organized into stem loops SLI, SLII, dumbbell structures DBI, DBII, a small hairpin (sHP) and a terminal 3’ stem loop (3’SL). Subgenomic RNA fragments (sfRNA) 1–4 are indicated. (B) Outline of RaPID assay. Plasmids expressing BoxB-flanked RNA and the λN-biotin ligase fusion protein gene (λN-HA-BirA) were co-transfected into mosquito cells. Subsequently, biotinylated proteins were captured using streptavidin beads and identified by LC-MS/MS. (C) Schematic of the EDEN15 RNA motifs (3 repeats of 15bp each) flanked by three BoxB RNA motifs each at their 5’ and 3’ ends. (D) Average fold change of proteins enriched in EDEN15 RNA expressing cells relative to the scrambled RNA control plotted against their SAINT probability scores. ELAV protein (shown in red) was enriched by ~40 fold (n = 2, **p
    Figure Legend Snippet: Diagram of the dengue viral (DENV) genome and strategy for the RNA-protein interaction (RAPID) assay. (A) DENV genome organization. The open reading frame encoding the three structural (C (capsid), prM/M (membrane), E (envelope)) proteins and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) proteins flanked by 5’ and 3’ untranslated regions (UTR) are shown. The 3’ UTR is organized into stem loops SLI, SLII, dumbbell structures DBI, DBII, a small hairpin (sHP) and a terminal 3’ stem loop (3’SL). Subgenomic RNA fragments (sfRNA) 1–4 are indicated. (B) Outline of RaPID assay. Plasmids expressing BoxB-flanked RNA and the λN-biotin ligase fusion protein gene (λN-HA-BirA) were co-transfected into mosquito cells. Subsequently, biotinylated proteins were captured using streptavidin beads and identified by LC-MS/MS. (C) Schematic of the EDEN15 RNA motifs (3 repeats of 15bp each) flanked by three BoxB RNA motifs each at their 5’ and 3’ ends. (D) Average fold change of proteins enriched in EDEN15 RNA expressing cells relative to the scrambled RNA control plotted against their SAINT probability scores. ELAV protein (shown in red) was enriched by ~40 fold (n = 2, **p

    Techniques Used: Expressing, Transfection, Liquid Chromatography with Mass Spectroscopy

    Colocalization and interaction of Loqs protein with DENV RNA. (A) Fluorescent in situ hybridization imaging of Aag2 cells infected with DENV2 at an MOI of 1 after 48 hrs. NS3 and Loqs proteins (shown in green) were visualized using labeled antibodies, while DENV RNA (shown in red) was visualized using labeled antisense RNA probes. Costes p value was calculated to measure the extent of colocalization of DENV2 RNA with NS3/Loqs proteins. (B) Immunoprecipitation of HA-tagged Loqs from Aag2 cells infected with DENV2 at a MOI of 1. Aag2 cells transfected with HA-GFP or HA-Loqs PA/PB plasmids were infected with DENV2 for 48 hrs, and immunoprecipitations were performed with anti-HA antibodies. Abundances of HA-GFP and HA-Loqs in input lysates and immunoprecipitated material measured by western blot analysis. (C) DENV2 and RPL32 RNA abundances in immunoprecipitated RNA (IP) and input RNA (10%) were measured by semi-quantitative RT-PCR. A representative agarose gel image from three independent experiments is shown. (D) DENV2 RNA abundance in immunoprecipitated RNA (IP) as measured by RT-qPCR. Data was normalized to RPL32 mRNA levels (n = 3, ***p = 0.0006).
    Figure Legend Snippet: Colocalization and interaction of Loqs protein with DENV RNA. (A) Fluorescent in situ hybridization imaging of Aag2 cells infected with DENV2 at an MOI of 1 after 48 hrs. NS3 and Loqs proteins (shown in green) were visualized using labeled antibodies, while DENV RNA (shown in red) was visualized using labeled antisense RNA probes. Costes p value was calculated to measure the extent of colocalization of DENV2 RNA with NS3/Loqs proteins. (B) Immunoprecipitation of HA-tagged Loqs from Aag2 cells infected with DENV2 at a MOI of 1. Aag2 cells transfected with HA-GFP or HA-Loqs PA/PB plasmids were infected with DENV2 for 48 hrs, and immunoprecipitations were performed with anti-HA antibodies. Abundances of HA-GFP and HA-Loqs in input lysates and immunoprecipitated material measured by western blot analysis. (C) DENV2 and RPL32 RNA abundances in immunoprecipitated RNA (IP) and input RNA (10%) were measured by semi-quantitative RT-PCR. A representative agarose gel image from three independent experiments is shown. (D) DENV2 RNA abundance in immunoprecipitated RNA (IP) as measured by RT-qPCR. Data was normalized to RPL32 mRNA levels (n = 3, ***p = 0.0006).

    Techniques Used: In Situ Hybridization, Imaging, Infection, Labeling, Immunoprecipitation, Transfection, Western Blot, Quantitative RT-PCR, Agarose Gel Electrophoresis

    Effects of Loqs depletion distinct RNA virus infections. Aag2 cells were infected with dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Zika virus (ZIKV) or chikungunya virus (CHIKV) at a MOI of 0.1 and harvested at 96 hrs post infection. Viral RNA abundances were measured by qPCR using specific primers. Data is represented as average fold-change over dsGFP from three independent experiments (*p
    Figure Legend Snippet: Effects of Loqs depletion distinct RNA virus infections. Aag2 cells were infected with dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Zika virus (ZIKV) or chikungunya virus (CHIKV) at a MOI of 0.1 and harvested at 96 hrs post infection. Viral RNA abundances were measured by qPCR using specific primers. Data is represented as average fold-change over dsGFP from three independent experiments (*p

    Techniques Used: Infection, Real-time Polymerase Chain Reaction

    Effects of Sec61A1 and Loquacious depletion on DENV2 RNA and protein abundances, and viral titers. (A) Experimental outline. Mosquito Aag2 cells were transfected with double stranded RNAs (dsRNA) directed against GFP, Sec61A1, Loqs (targeting both PA and PB isoforms) or Loqs-PB mRNAs. 24 hrs post transfection, cells were infected with DENV2-NGC at an MOI of 0.1 and harvested 96 hrs post infection for analyses. (B) RT-qPCR measurement of DENV RNA abundances in dsRNA-treated cells plotted as fold change over treatment with dsGFP. Data was normalized to internal control RPL32 mRNA levels (n = 3, ****p
    Figure Legend Snippet: Effects of Sec61A1 and Loquacious depletion on DENV2 RNA and protein abundances, and viral titers. (A) Experimental outline. Mosquito Aag2 cells were transfected with double stranded RNAs (dsRNA) directed against GFP, Sec61A1, Loqs (targeting both PA and PB isoforms) or Loqs-PB mRNAs. 24 hrs post transfection, cells were infected with DENV2-NGC at an MOI of 0.1 and harvested 96 hrs post infection for analyses. (B) RT-qPCR measurement of DENV RNA abundances in dsRNA-treated cells plotted as fold change over treatment with dsGFP. Data was normalized to internal control RPL32 mRNA levels (n = 3, ****p

    Techniques Used: Transfection, Infection, Quantitative RT-PCR

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    New England Biolabs m7 gpppa rna cap structure analog
    Structural and mutagenesis analysis of RNase AM activities. (A) Overall crystal <t>structure</t> of RNase AM PHP domain (teal) with manganese ions (purple) and sulfate (yellow and red) shown as spheres. (B) Overlay of RNase AM crystal structure (teal) with RNase AM from AlphaFold (light blue) showing the insertion domain. (C) Overlay of RNase AM with CV1693 (gray) with AMP from CV1693 shown as sticks. (D) The active site of RNase AM showing the coordination of the three manganese ions and sulfate to the active site residues (sticks). AMP and manganese (gray) and the phosphate (orange and red) from the overlay with CV1693 are shown. (E) Electrostatic potential mapped onto the surface of RNase AM showing the positively charged region where the 5’P from CV1693 AMP (gray) is buried. (F) Comparison of binding of AMP (gray) between RNase AM and CV1693 showing conservation of the residues that interact with AMP. (G) Reaction products of in vitro deFADding assays with 100 nM recombinant RNase AM and putative catalytically inactive point mutants. Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped <t>RNA</t> (FAD–) are indicated. The asterisk represents the 32 P-labeling within the body of the RNA. Reaction products were resolved on 15% 7M urea PAGE gels
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    Structural and mutagenesis analysis of RNase AM activities. (A) Overall crystal structure of RNase AM PHP domain (teal) with manganese ions (purple) and sulfate (yellow and red) shown as spheres. (B) Overlay of RNase AM crystal structure (teal) with RNase AM from AlphaFold (light blue) showing the insertion domain. (C) Overlay of RNase AM with CV1693 (gray) with AMP from CV1693 shown as sticks. (D) The active site of RNase AM showing the coordination of the three manganese ions and sulfate to the active site residues (sticks). AMP and manganese (gray) and the phosphate (orange and red) from the overlay with CV1693 are shown. (E) Electrostatic potential mapped onto the surface of RNase AM showing the positively charged region where the 5’P from CV1693 AMP (gray) is buried. (F) Comparison of binding of AMP (gray) between RNase AM and CV1693 showing conservation of the residues that interact with AMP. (G) Reaction products of in vitro deFADding assays with 100 nM recombinant RNase AM and putative catalytically inactive point mutants. Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped RNA (FAD–) are indicated. The asterisk represents the 32 P-labeling within the body of the RNA. Reaction products were resolved on 15% 7M urea PAGE gels

    Journal: bioRxiv

    Article Title: Identification of a novel deFADding activity in 5’ to 3’ exoribonucleases

    doi: 10.1101/2022.05.10.491372

    Figure Lengend Snippet: Structural and mutagenesis analysis of RNase AM activities. (A) Overall crystal structure of RNase AM PHP domain (teal) with manganese ions (purple) and sulfate (yellow and red) shown as spheres. (B) Overlay of RNase AM crystal structure (teal) with RNase AM from AlphaFold (light blue) showing the insertion domain. (C) Overlay of RNase AM with CV1693 (gray) with AMP from CV1693 shown as sticks. (D) The active site of RNase AM showing the coordination of the three manganese ions and sulfate to the active site residues (sticks). AMP and manganese (gray) and the phosphate (orange and red) from the overlay with CV1693 are shown. (E) Electrostatic potential mapped onto the surface of RNase AM showing the positively charged region where the 5’P from CV1693 AMP (gray) is buried. (F) Comparison of binding of AMP (gray) between RNase AM and CV1693 showing conservation of the residues that interact with AMP. (G) Reaction products of in vitro deFADding assays with 100 nM recombinant RNase AM and putative catalytically inactive point mutants. Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped RNA (FAD–) are indicated. The asterisk represents the 32 P-labeling within the body of the RNA. Reaction products were resolved on 15% 7M urea PAGE gels

    Article Snippet: For m7 G-capped RNA, m7 GpppA RNA Cap Structure Analog (New England Biolabs) was included in the transcription reaction.

    Techniques: Mutagenesis, Binding Assay, In Vitro, Recombinant, Labeling, Polyacrylamide Gel Electrophoresis

    Xrn1 deFADs FAD-capped RNAs in both yeast and human cells. (A) Time-course decay analysis of uniformly 32 P-labeled m 7 G- or FAD-capped RNA in the presence of cell extract prepared from WT or xrn1Δ strains. The remaining RNA was quantified and plotted from three independent experiments with ± SD denoted by error bars. (B) FAD-capQ assay using different amounts of in vitro transcribed FAD-capped RNA to demonstrate the release of intact FAD upon Xrn1 mediated deFADding. Catalytic-dead mutant of Xrn1, E178Q was used as a control for the Xrn1 reaction and NAD-capped RNA was used as a control for specific detection of FAD in the assay (C) Total RNAs from WT and xrn1Δ were subjected to the FAD-capQ assay to detect total levels of cellular FAD-capped RNA. Data represents average from three independent experiments. Error bars represent ± SD with unpaired t -test; * p

    Journal: bioRxiv

    Article Title: Identification of a novel deFADding activity in 5’ to 3’ exoribonucleases

    doi: 10.1101/2022.05.10.491372

    Figure Lengend Snippet: Xrn1 deFADs FAD-capped RNAs in both yeast and human cells. (A) Time-course decay analysis of uniformly 32 P-labeled m 7 G- or FAD-capped RNA in the presence of cell extract prepared from WT or xrn1Δ strains. The remaining RNA was quantified and plotted from three independent experiments with ± SD denoted by error bars. (B) FAD-capQ assay using different amounts of in vitro transcribed FAD-capped RNA to demonstrate the release of intact FAD upon Xrn1 mediated deFADding. Catalytic-dead mutant of Xrn1, E178Q was used as a control for the Xrn1 reaction and NAD-capped RNA was used as a control for specific detection of FAD in the assay (C) Total RNAs from WT and xrn1Δ were subjected to the FAD-capQ assay to detect total levels of cellular FAD-capped RNA. Data represents average from three independent experiments. Error bars represent ± SD with unpaired t -test; * p

    Article Snippet: For m7 G-capped RNA, m7 GpppA RNA Cap Structure Analog (New England Biolabs) was included in the transcription reaction.

    Techniques: Labeling, In Vitro, Mutagenesis

    RNase AM is a novel deFADding enzyme in E. coli. (A) Reaction products of in vitro deFADding assays with 100nM recombinant RNase AM. Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped RNA (FAD–), NAD-capped (NAD–), and m 7 G-capped (m 7 G–). The asterisk represents the 32 P-labeling within the body of the RNA. Reaction products were resolved on 15% 7M urea PAGE gels. (B) Time-course decay analysis of uniformly 32 P-labeled monophosphate or FAD-capped RNA with the indicated amount of RNase AM protein are shown. Quantitation of RNA remaining is plotted from three independent experiments with ± SD denoted by error bars. (C) To demonstrate that RNase AM releases intact FAD upon deFADding like Xrn1 (in Fig 3 ), FAD-capQ assay using different amount of in vitro transcribed FAD-capped RNA was used. Catalytic-dead mutant of RNase AM, D20A was used as control for the reaction. (D) Total RNA from WT and rnase AMΔ were subjected to the FAD-capQ assay to detect total levels of cellular FAD-capped RNA. Data represents an average from three independent experiments. Error bars represent ± SD with unpaired t -test; * p

    Journal: bioRxiv

    Article Title: Identification of a novel deFADding activity in 5’ to 3’ exoribonucleases

    doi: 10.1101/2022.05.10.491372

    Figure Lengend Snippet: RNase AM is a novel deFADding enzyme in E. coli. (A) Reaction products of in vitro deFADding assays with 100nM recombinant RNase AM. Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped RNA (FAD–), NAD-capped (NAD–), and m 7 G-capped (m 7 G–). The asterisk represents the 32 P-labeling within the body of the RNA. Reaction products were resolved on 15% 7M urea PAGE gels. (B) Time-course decay analysis of uniformly 32 P-labeled monophosphate or FAD-capped RNA with the indicated amount of RNase AM protein are shown. Quantitation of RNA remaining is plotted from three independent experiments with ± SD denoted by error bars. (C) To demonstrate that RNase AM releases intact FAD upon deFADding like Xrn1 (in Fig 3 ), FAD-capQ assay using different amount of in vitro transcribed FAD-capped RNA was used. Catalytic-dead mutant of RNase AM, D20A was used as control for the reaction. (D) Total RNA from WT and rnase AMΔ were subjected to the FAD-capQ assay to detect total levels of cellular FAD-capped RNA. Data represents an average from three independent experiments. Error bars represent ± SD with unpaired t -test; * p

    Article Snippet: For m7 G-capped RNA, m7 GpppA RNA Cap Structure Analog (New England Biolabs) was included in the transcription reaction.

    Techniques: In Vitro, Recombinant, Labeling, Polyacrylamide Gel Electrophoresis, Quantitation Assay, Mutagenesis

    Identification of FAD cap binding proteins in budding yeast. (A) Schematic illustration of the FAD cap -RNA Affinity Purification (FcRAP). (B) Eluates from FcRAP were loaded on to a 4%-12% Bis-Tris gel and stained with SYPRO Ruby. The m 7 G cap affinity purification was used as a control. All protein bands labeled on the gel were excised from the gel and identified using mass spectrometry. (C) Electrophoretic mobility shift assay (EMSA) using increasing concentration of Xrn1 (2.5 pmol, 5 pmol and 7.5 pmol) and a fixed concentration (5 pmol) of uniformly labeled in vitro transcribed FADcapped or NAD-capped or triphosphate RNA (pppA–). (D) EMSA using equimolar concentration (5pmol) of Xrn1 and uniformly labeled in vitro transcribed FAD-capped or NAD-capped or pppA–RNAs in the presence of total yeast RNA as a nonspecific competitor.

    Journal: bioRxiv

    Article Title: Identification of a novel deFADding activity in 5’ to 3’ exoribonucleases

    doi: 10.1101/2022.05.10.491372

    Figure Lengend Snippet: Identification of FAD cap binding proteins in budding yeast. (A) Schematic illustration of the FAD cap -RNA Affinity Purification (FcRAP). (B) Eluates from FcRAP were loaded on to a 4%-12% Bis-Tris gel and stained with SYPRO Ruby. The m 7 G cap affinity purification was used as a control. All protein bands labeled on the gel were excised from the gel and identified using mass spectrometry. (C) Electrophoretic mobility shift assay (EMSA) using increasing concentration of Xrn1 (2.5 pmol, 5 pmol and 7.5 pmol) and a fixed concentration (5 pmol) of uniformly labeled in vitro transcribed FADcapped or NAD-capped or triphosphate RNA (pppA–). (D) EMSA using equimolar concentration (5pmol) of Xrn1 and uniformly labeled in vitro transcribed FAD-capped or NAD-capped or pppA–RNAs in the presence of total yeast RNA as a nonspecific competitor.

    Article Snippet: For m7 G-capped RNA, m7 GpppA RNA Cap Structure Analog (New England Biolabs) was included in the transcription reaction.

    Techniques: Binding Assay, Affinity Purification, Staining, Labeling, Mass Spectrometry, Electrophoretic Mobility Shift Assay, Concentration Assay, In Vitro

    Xrn1 and Rat1 are deFADding enzymes. (A) Chemical structure of FAD-capped RNA. (B) Reaction products of in vitro deFADding assays with 50 nM recombinant Xrn1, WT, and catalytically inactive ( E178Q ) from K. lactis or 100 nM recombinant Rat1, WT or catalytically inactive ( E207Q ) from S. pombe or 25 nM Rai1, WT catalytically inactive ( D201A) (S. pombe ). Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped RNA (FAD–) where the line denotes the RNA were used as indicated. The asterisk represents the 32 P-labeling within the body of the RNA. Reaction products were resolved on 15% 7M urea PAGE gels. Time-course decay analysis of uniformly 32 P-labeled monophosphate or FAD-capped RNA with the indicated amount of Xrn1 (C) or Rat1 (D) protein are shown. Quantitation of RNA remaining is plotted from three independent experiments with ± SD denoted by error bars.

    Journal: bioRxiv

    Article Title: Identification of a novel deFADding activity in 5’ to 3’ exoribonucleases

    doi: 10.1101/2022.05.10.491372

    Figure Lengend Snippet: Xrn1 and Rat1 are deFADding enzymes. (A) Chemical structure of FAD-capped RNA. (B) Reaction products of in vitro deFADding assays with 50 nM recombinant Xrn1, WT, and catalytically inactive ( E178Q ) from K. lactis or 100 nM recombinant Rat1, WT or catalytically inactive ( E207Q ) from S. pombe or 25 nM Rai1, WT catalytically inactive ( D201A) (S. pombe ). Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped RNA (FAD–) where the line denotes the RNA were used as indicated. The asterisk represents the 32 P-labeling within the body of the RNA. Reaction products were resolved on 15% 7M urea PAGE gels. Time-course decay analysis of uniformly 32 P-labeled monophosphate or FAD-capped RNA with the indicated amount of Xrn1 (C) or Rat1 (D) protein are shown. Quantitation of RNA remaining is plotted from three independent experiments with ± SD denoted by error bars.

    Article Snippet: For m7 G-capped RNA, m7 GpppA RNA Cap Structure Analog (New England Biolabs) was included in the transcription reaction.

    Techniques: In Vitro, Recombinant, Labeling, Polyacrylamide Gel Electrophoresis, Quantitation Assay

    K m values for substrates of nsp14 a) Titration of various nsp14 susbstrates. GTP, GpppA cap analogue, GpppA-RNA, and me 7 GpppA cap analogue were incubated for 20 minutes with 5 nM nsp14 in the presence of 1 μM SAM. b-d) Determination of Michaelis constants for ( b ) GpppA-RNA, ( c ) GpppA, and ( d ) GTP for nsp14. nsp14 concentration was fixed at 5 nM for all experiments, and substrate concentration varied. V max values cannot be compared between substrates, and are therefore not given. HTRF values are given as raw, and not normalised, HTRF values. Errors given are 95% confidence ranges.

    Journal: bioRxiv

    Article Title: Identification of SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of the nsp14 RNA Cap Methyltransferase

    doi: 10.1101/2021.04.07.438810

    Figure Lengend Snippet: K m values for substrates of nsp14 a) Titration of various nsp14 susbstrates. GTP, GpppA cap analogue, GpppA-RNA, and me 7 GpppA cap analogue were incubated for 20 minutes with 5 nM nsp14 in the presence of 1 μM SAM. b-d) Determination of Michaelis constants for ( b ) GpppA-RNA, ( c ) GpppA, and ( d ) GTP for nsp14. nsp14 concentration was fixed at 5 nM for all experiments, and substrate concentration varied. V max values cannot be compared between substrates, and are therefore not given. HTRF values are given as raw, and not normalised, HTRF values. Errors given are 95% confidence ranges.

    Article Snippet: For screening, the methyltransferase reaction was conducted at room temperature in an 8 μl reaction volume with 10 nM nsp14, 1 μM Ultrapure SAM (CisBio), 0.14 mM GpppA RNA cap analogue (New England Biolabs) in reaction buffer consisting of HEPES-KOH pH 7.6, 150 mM NaCl, and 0.5 mM DTT.

    Techniques: Titration, Incubation, Concentration Assay

    Nsp14 inhibitors do not inhibit nsp10-16 a) Gel filtration from final purification step of the nsp10-16 fusion protein. Coomassie shows fractions taken across the major peak of the gel filtration elution. Only fractions indicated were pooled (black bar, upper). Expected size of nsp10-16: 47.8 kDa (nsp10 - 13.3 kDa + nsp16 - kDa 34.5 kDa) b) Time course of the nsp10-16 methyltransferase reaction with 100 nM, 50 nM, 25 nM and 12.5 nM nsp10-16 enzyme. Reaction was conducted with 1.3 μM me 7 GpppA-RNA and 1 μM SAM. c) Determination of Michaelis constants for me 7 GpppA-RNA for nsp10-16. nsp10-16 concentration was fixed at 100 nM and substrate concentration varied. Errors given are 95% confidence ranges. d) Cross validation of nsp14 inhibitors with nsp10-16. Normalised HTRF values for nsp10-16 with inhibitors identified for nsp14 and sinefungin. All compounds tested at 50 μM. Reactions conducted with 1.3 μM me 7 GpppA-RNA and 1 μM SAM.

    Journal: bioRxiv

    Article Title: Identification of SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of the nsp14 RNA Cap Methyltransferase

    doi: 10.1101/2021.04.07.438810

    Figure Lengend Snippet: Nsp14 inhibitors do not inhibit nsp10-16 a) Gel filtration from final purification step of the nsp10-16 fusion protein. Coomassie shows fractions taken across the major peak of the gel filtration elution. Only fractions indicated were pooled (black bar, upper). Expected size of nsp10-16: 47.8 kDa (nsp10 - 13.3 kDa + nsp16 - kDa 34.5 kDa) b) Time course of the nsp10-16 methyltransferase reaction with 100 nM, 50 nM, 25 nM and 12.5 nM nsp10-16 enzyme. Reaction was conducted with 1.3 μM me 7 GpppA-RNA and 1 μM SAM. c) Determination of Michaelis constants for me 7 GpppA-RNA for nsp10-16. nsp10-16 concentration was fixed at 100 nM and substrate concentration varied. Errors given are 95% confidence ranges. d) Cross validation of nsp14 inhibitors with nsp10-16. Normalised HTRF values for nsp10-16 with inhibitors identified for nsp14 and sinefungin. All compounds tested at 50 μM. Reactions conducted with 1.3 μM me 7 GpppA-RNA and 1 μM SAM.

    Article Snippet: For screening, the methyltransferase reaction was conducted at room temperature in an 8 μl reaction volume with 10 nM nsp14, 1 μM Ultrapure SAM (CisBio), 0.14 mM GpppA RNA cap analogue (New England Biolabs) in reaction buffer consisting of HEPES-KOH pH 7.6, 150 mM NaCl, and 0.5 mM DTT.

    Techniques: Filtration, Purification, Concentration Assay

    An HTRF based assay for methyltransferase activity a) Outline of the HTRF based assay for methyltransferase activity. Both nsp14 and nsp10-16 are SAM dependent methyltransferases that produce SAH following successful methyltransfer to their substrate. This SAH displaces SAH-d2 from the variable region of an α-SAH Tb cryptate-conjugated antibody, thus lowering HTRF signal through the disruption of the Tb cryptate – d2 FRET pair. b) nsp14 was assayed for methyltransferase activity through the HTRF based assay. The methyltransferase reaction was run in either the absence of 10nM nsp14, 1 μM SAM, 0.11 mM GpppA-RNA, or in the presence of all three components. In addition, the methyltransferase reaction was conducted in the presence of 0.25 μM, 2.5 μM and 25 μM of the pan-methyltransferase inhibitor Sinefungin, which acts as a competitive inhibitor (with respect to SAM) towards SAM-dependent methyltransferases. c) Time course of nsp14 activity by HTRF assay. 20 nM of nsp14 was incubated with 0.11 mM GpppA cap analogue and 1 μM SAM for the time indicated. In addition, an experiment was run in the absence of nsp14, and in the presence of the methyltransferase inhibitor sinefungin. The reaction was started as a master mix with the addition of nsp14, and 8 μl was removed at every time point and added to 2 μl of 5M NaCl to stop the methyltransferase reaction. Points are the mean of three technical repeats, and error bars indicate range.

    Journal: bioRxiv

    Article Title: Identification of SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of the nsp14 RNA Cap Methyltransferase

    doi: 10.1101/2021.04.07.438810

    Figure Lengend Snippet: An HTRF based assay for methyltransferase activity a) Outline of the HTRF based assay for methyltransferase activity. Both nsp14 and nsp10-16 are SAM dependent methyltransferases that produce SAH following successful methyltransfer to their substrate. This SAH displaces SAH-d2 from the variable region of an α-SAH Tb cryptate-conjugated antibody, thus lowering HTRF signal through the disruption of the Tb cryptate – d2 FRET pair. b) nsp14 was assayed for methyltransferase activity through the HTRF based assay. The methyltransferase reaction was run in either the absence of 10nM nsp14, 1 μM SAM, 0.11 mM GpppA-RNA, or in the presence of all three components. In addition, the methyltransferase reaction was conducted in the presence of 0.25 μM, 2.5 μM and 25 μM of the pan-methyltransferase inhibitor Sinefungin, which acts as a competitive inhibitor (with respect to SAM) towards SAM-dependent methyltransferases. c) Time course of nsp14 activity by HTRF assay. 20 nM of nsp14 was incubated with 0.11 mM GpppA cap analogue and 1 μM SAM for the time indicated. In addition, an experiment was run in the absence of nsp14, and in the presence of the methyltransferase inhibitor sinefungin. The reaction was started as a master mix with the addition of nsp14, and 8 μl was removed at every time point and added to 2 μl of 5M NaCl to stop the methyltransferase reaction. Points are the mean of three technical repeats, and error bars indicate range.

    Article Snippet: For screening, the methyltransferase reaction was conducted at room temperature in an 8 μl reaction volume with 10 nM nsp14, 1 μM Ultrapure SAM (CisBio), 0.14 mM GpppA RNA cap analogue (New England Biolabs) in reaction buffer consisting of HEPES-KOH pH 7.6, 150 mM NaCl, and 0.5 mM DTT.

    Techniques: HTRF Assay, Activity Assay, Incubation

    Effect of T3 on THRβ mRNA expression in the periventricular zone of zebrafish embryonic brain. Embryos were exposed from 4- to 22- hpf to T3. Note the prominent blue coloration in control, ( A ) localized to fore- and midbrain regions (red arrow). By comparison, panels B,C and E show less blue coloration (red arrows) upon exposure to a range of T3 from 0.1 nM to 100 nM. The average intensity of THRβ expression recorded in brain at 22 somite stage showed dose dependent reduction (histogram, E ). ( F ) Bar chart of THRβ expression based on quantitative PCR (qRT-PCR, total RNA from whole embryos) at 22 somite stage. The 10 and 100 nM T3 was significantly different from the control (*, P

    Journal: Endocrine disruptors (Austin, Tex.)

    Article Title: The PBDE metabolite 6-OH-BDE 47 affects melanin pigmentation and THRβ MRNA expression in the eye of zebrafish embryos

    doi: 10.4161/23273739.2014.969072

    Figure Lengend Snippet: Effect of T3 on THRβ mRNA expression in the periventricular zone of zebrafish embryonic brain. Embryos were exposed from 4- to 22- hpf to T3. Note the prominent blue coloration in control, ( A ) localized to fore- and midbrain regions (red arrow). By comparison, panels B,C and E show less blue coloration (red arrows) upon exposure to a range of T3 from 0.1 nM to 100 nM. The average intensity of THRβ expression recorded in brain at 22 somite stage showed dose dependent reduction (histogram, E ). ( F ) Bar chart of THRβ expression based on quantitative PCR (qRT-PCR, total RNA from whole embryos) at 22 somite stage. The 10 and 100 nM T3 was significantly different from the control (*, P

    Article Snippet: THRβ mRNA was synthesized with SP6 polymerase and capped using a G(5’)ppp(5’)A RNA cap structure analog (New England Biolabs).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Effect of Loqs depletion on DENV RNA translation, replication and stability. (A) Western blot analysis of NS3, Loqs and GAPDH protein abundances in cytosolic and ER membrane fractions isolated from DENV2-infected Aag2 cell lysates at 72 hrs post infection. Representative image from three independent experiments is shown. (B) Immunoprecipitation of HA-tagged Loqs from Aag2 cells infected with DENV2 at a MOI of 1. Aag2 cells transfected with HA-GFP or HA-Loqs PA/PB plasmids were infected with DENV2 for 72 hrs and immunoprecipitations were performed with anti-HA antibody with or without RNaseA/T1 treatment. The abundances of DENV NS3, NS4B and capsid proteins in the immunoprecipitated material (IP) and the input lysates (10%) were determined by western blot analysis. (C) Luciferase activities of wildtype and replication-defective DENV2 luciferase replicons in C6/36 cells transfected with control siRNAs or siRNAs against Loqs (siLoqs-4 and siLoqs-5 were used at a final concentration of 25nM each). C6/36 cells were transfected with the indicated siRNAs followed by wildtype or replication-defective (NS5-GDD) replicon RNAs and harvested at the indicated time points. Average luciferase expression from DENV replicons from six independent replicates is shown (***p

    Journal: PLoS Pathogens

    Article Title: Loquacious modulates flaviviral RNA replication in mosquito cells

    doi: 10.1371/journal.ppat.1010163

    Figure Lengend Snippet: Effect of Loqs depletion on DENV RNA translation, replication and stability. (A) Western blot analysis of NS3, Loqs and GAPDH protein abundances in cytosolic and ER membrane fractions isolated from DENV2-infected Aag2 cell lysates at 72 hrs post infection. Representative image from three independent experiments is shown. (B) Immunoprecipitation of HA-tagged Loqs from Aag2 cells infected with DENV2 at a MOI of 1. Aag2 cells transfected with HA-GFP or HA-Loqs PA/PB plasmids were infected with DENV2 for 72 hrs and immunoprecipitations were performed with anti-HA antibody with or without RNaseA/T1 treatment. The abundances of DENV NS3, NS4B and capsid proteins in the immunoprecipitated material (IP) and the input lysates (10%) were determined by western blot analysis. (C) Luciferase activities of wildtype and replication-defective DENV2 luciferase replicons in C6/36 cells transfected with control siRNAs or siRNAs against Loqs (siLoqs-4 and siLoqs-5 were used at a final concentration of 25nM each). C6/36 cells were transfected with the indicated siRNAs followed by wildtype or replication-defective (NS5-GDD) replicon RNAs and harvested at the indicated time points. Average luciferase expression from DENV replicons from six independent replicates is shown (***p

    Article Snippet: 5μg of the XbaI-linearized plasmid was incubated with 1.3 μl of 75mM rATP, 6.7 μl each of 75mM rCTP, rGTP and rUTP, 10 μl of 10X reaction buffer, 10 μl of T7 enzyme mix and 12.5 μl of 5’GpppA cap analog (S1406S, NEB) in a final reaction volume of 100 μl for 30 min at 30°C.

    Techniques: Western Blot, Isolation, Infection, Immunoprecipitation, Transfection, Luciferase, Concentration Assay, Expressing

    Diagram of the dengue viral (DENV) genome and strategy for the RNA-protein interaction (RAPID) assay. (A) DENV genome organization. The open reading frame encoding the three structural (C (capsid), prM/M (membrane), E (envelope)) proteins and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) proteins flanked by 5’ and 3’ untranslated regions (UTR) are shown. The 3’ UTR is organized into stem loops SLI, SLII, dumbbell structures DBI, DBII, a small hairpin (sHP) and a terminal 3’ stem loop (3’SL). Subgenomic RNA fragments (sfRNA) 1–4 are indicated. (B) Outline of RaPID assay. Plasmids expressing BoxB-flanked RNA and the λN-biotin ligase fusion protein gene (λN-HA-BirA) were co-transfected into mosquito cells. Subsequently, biotinylated proteins were captured using streptavidin beads and identified by LC-MS/MS. (C) Schematic of the EDEN15 RNA motifs (3 repeats of 15bp each) flanked by three BoxB RNA motifs each at their 5’ and 3’ ends. (D) Average fold change of proteins enriched in EDEN15 RNA expressing cells relative to the scrambled RNA control plotted against their SAINT probability scores. ELAV protein (shown in red) was enriched by ~40 fold (n = 2, **p

    Journal: PLoS Pathogens

    Article Title: Loquacious modulates flaviviral RNA replication in mosquito cells

    doi: 10.1371/journal.ppat.1010163

    Figure Lengend Snippet: Diagram of the dengue viral (DENV) genome and strategy for the RNA-protein interaction (RAPID) assay. (A) DENV genome organization. The open reading frame encoding the three structural (C (capsid), prM/M (membrane), E (envelope)) proteins and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) proteins flanked by 5’ and 3’ untranslated regions (UTR) are shown. The 3’ UTR is organized into stem loops SLI, SLII, dumbbell structures DBI, DBII, a small hairpin (sHP) and a terminal 3’ stem loop (3’SL). Subgenomic RNA fragments (sfRNA) 1–4 are indicated. (B) Outline of RaPID assay. Plasmids expressing BoxB-flanked RNA and the λN-biotin ligase fusion protein gene (λN-HA-BirA) were co-transfected into mosquito cells. Subsequently, biotinylated proteins were captured using streptavidin beads and identified by LC-MS/MS. (C) Schematic of the EDEN15 RNA motifs (3 repeats of 15bp each) flanked by three BoxB RNA motifs each at their 5’ and 3’ ends. (D) Average fold change of proteins enriched in EDEN15 RNA expressing cells relative to the scrambled RNA control plotted against their SAINT probability scores. ELAV protein (shown in red) was enriched by ~40 fold (n = 2, **p

    Article Snippet: 5μg of the XbaI-linearized plasmid was incubated with 1.3 μl of 75mM rATP, 6.7 μl each of 75mM rCTP, rGTP and rUTP, 10 μl of 10X reaction buffer, 10 μl of T7 enzyme mix and 12.5 μl of 5’GpppA cap analog (S1406S, NEB) in a final reaction volume of 100 μl for 30 min at 30°C.

    Techniques: Expressing, Transfection, Liquid Chromatography with Mass Spectroscopy

    Colocalization and interaction of Loqs protein with DENV RNA. (A) Fluorescent in situ hybridization imaging of Aag2 cells infected with DENV2 at an MOI of 1 after 48 hrs. NS3 and Loqs proteins (shown in green) were visualized using labeled antibodies, while DENV RNA (shown in red) was visualized using labeled antisense RNA probes. Costes p value was calculated to measure the extent of colocalization of DENV2 RNA with NS3/Loqs proteins. (B) Immunoprecipitation of HA-tagged Loqs from Aag2 cells infected with DENV2 at a MOI of 1. Aag2 cells transfected with HA-GFP or HA-Loqs PA/PB plasmids were infected with DENV2 for 48 hrs, and immunoprecipitations were performed with anti-HA antibodies. Abundances of HA-GFP and HA-Loqs in input lysates and immunoprecipitated material measured by western blot analysis. (C) DENV2 and RPL32 RNA abundances in immunoprecipitated RNA (IP) and input RNA (10%) were measured by semi-quantitative RT-PCR. A representative agarose gel image from three independent experiments is shown. (D) DENV2 RNA abundance in immunoprecipitated RNA (IP) as measured by RT-qPCR. Data was normalized to RPL32 mRNA levels (n = 3, ***p = 0.0006).

    Journal: PLoS Pathogens

    Article Title: Loquacious modulates flaviviral RNA replication in mosquito cells

    doi: 10.1371/journal.ppat.1010163

    Figure Lengend Snippet: Colocalization and interaction of Loqs protein with DENV RNA. (A) Fluorescent in situ hybridization imaging of Aag2 cells infected with DENV2 at an MOI of 1 after 48 hrs. NS3 and Loqs proteins (shown in green) were visualized using labeled antibodies, while DENV RNA (shown in red) was visualized using labeled antisense RNA probes. Costes p value was calculated to measure the extent of colocalization of DENV2 RNA with NS3/Loqs proteins. (B) Immunoprecipitation of HA-tagged Loqs from Aag2 cells infected with DENV2 at a MOI of 1. Aag2 cells transfected with HA-GFP or HA-Loqs PA/PB plasmids were infected with DENV2 for 48 hrs, and immunoprecipitations were performed with anti-HA antibodies. Abundances of HA-GFP and HA-Loqs in input lysates and immunoprecipitated material measured by western blot analysis. (C) DENV2 and RPL32 RNA abundances in immunoprecipitated RNA (IP) and input RNA (10%) were measured by semi-quantitative RT-PCR. A representative agarose gel image from three independent experiments is shown. (D) DENV2 RNA abundance in immunoprecipitated RNA (IP) as measured by RT-qPCR. Data was normalized to RPL32 mRNA levels (n = 3, ***p = 0.0006).

    Article Snippet: 5μg of the XbaI-linearized plasmid was incubated with 1.3 μl of 75mM rATP, 6.7 μl each of 75mM rCTP, rGTP and rUTP, 10 μl of 10X reaction buffer, 10 μl of T7 enzyme mix and 12.5 μl of 5’GpppA cap analog (S1406S, NEB) in a final reaction volume of 100 μl for 30 min at 30°C.

    Techniques: In Situ Hybridization, Imaging, Infection, Labeling, Immunoprecipitation, Transfection, Western Blot, Quantitative RT-PCR, Agarose Gel Electrophoresis

    Effects of Loqs depletion distinct RNA virus infections. Aag2 cells were infected with dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Zika virus (ZIKV) or chikungunya virus (CHIKV) at a MOI of 0.1 and harvested at 96 hrs post infection. Viral RNA abundances were measured by qPCR using specific primers. Data is represented as average fold-change over dsGFP from three independent experiments (*p

    Journal: PLoS Pathogens

    Article Title: Loquacious modulates flaviviral RNA replication in mosquito cells

    doi: 10.1371/journal.ppat.1010163

    Figure Lengend Snippet: Effects of Loqs depletion distinct RNA virus infections. Aag2 cells were infected with dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Zika virus (ZIKV) or chikungunya virus (CHIKV) at a MOI of 0.1 and harvested at 96 hrs post infection. Viral RNA abundances were measured by qPCR using specific primers. Data is represented as average fold-change over dsGFP from three independent experiments (*p

    Article Snippet: 5μg of the XbaI-linearized plasmid was incubated with 1.3 μl of 75mM rATP, 6.7 μl each of 75mM rCTP, rGTP and rUTP, 10 μl of 10X reaction buffer, 10 μl of T7 enzyme mix and 12.5 μl of 5’GpppA cap analog (S1406S, NEB) in a final reaction volume of 100 μl for 30 min at 30°C.

    Techniques: Infection, Real-time Polymerase Chain Reaction

    Effects of Sec61A1 and Loquacious depletion on DENV2 RNA and protein abundances, and viral titers. (A) Experimental outline. Mosquito Aag2 cells were transfected with double stranded RNAs (dsRNA) directed against GFP, Sec61A1, Loqs (targeting both PA and PB isoforms) or Loqs-PB mRNAs. 24 hrs post transfection, cells were infected with DENV2-NGC at an MOI of 0.1 and harvested 96 hrs post infection for analyses. (B) RT-qPCR measurement of DENV RNA abundances in dsRNA-treated cells plotted as fold change over treatment with dsGFP. Data was normalized to internal control RPL32 mRNA levels (n = 3, ****p

    Journal: PLoS Pathogens

    Article Title: Loquacious modulates flaviviral RNA replication in mosquito cells

    doi: 10.1371/journal.ppat.1010163

    Figure Lengend Snippet: Effects of Sec61A1 and Loquacious depletion on DENV2 RNA and protein abundances, and viral titers. (A) Experimental outline. Mosquito Aag2 cells were transfected with double stranded RNAs (dsRNA) directed against GFP, Sec61A1, Loqs (targeting both PA and PB isoforms) or Loqs-PB mRNAs. 24 hrs post transfection, cells were infected with DENV2-NGC at an MOI of 0.1 and harvested 96 hrs post infection for analyses. (B) RT-qPCR measurement of DENV RNA abundances in dsRNA-treated cells plotted as fold change over treatment with dsGFP. Data was normalized to internal control RPL32 mRNA levels (n = 3, ****p

    Article Snippet: 5μg of the XbaI-linearized plasmid was incubated with 1.3 μl of 75mM rATP, 6.7 μl each of 75mM rCTP, rGTP and rUTP, 10 μl of 10X reaction buffer, 10 μl of T7 enzyme mix and 12.5 μl of 5’GpppA cap analog (S1406S, NEB) in a final reaction volume of 100 μl for 30 min at 30°C.

    Techniques: Transfection, Infection, Quantitative RT-PCR