Journal: Nucleic Acids Research

Article Title: A Myc–microRNA network promotes exit from quiescence by suppressing the interferon response and cell-cycle arrest genes

doi: 10.1093/nar/gks1452

Figure Lengend Snippet: miR-22 targets genes mediating cell-cycle arrest. ( A ) Ago2 IP. Ago2 forms a complex with the miRNA and the target mRNA. This complex is immunoprecipitated and the mRNA levels are quantified with qRT-PCR. ( B ) Ago2 IPs identify genes mediating cell-cycle arrest and apoptosis as direct targets of miR-22. REDD1, TP53INP1, p21 and CARF transcripts show significantly higher Ago2 occupancy in miR-22–transfected HeLa cells compared with mock-transfected cells. Ago2 occupancy of the target genes transcripts was measured using qRT-PCR as described in the text. The Y -axis shows fold change in mRNA levels from Ago2 IP–isolated RNA normalized to input RNA, which served as the control. ( C ) Luciferase assays show that miR-22 directly targets the 3 ′ UTRs of the genes indicated on the X -axis. IKKg and Empty are negative controls. IKKg was not repressed by miR-22 and Empty is the luciferase vector with no UTR. The Y -axis shows relative luciferase units from miR-22-transfected cells normalized to control siRNA transfection. For the mutant 3′UTRs, 3 bp in each 6-mer miR-22 target site in the 3′UTRs were mutated. ( D ) qRT-PCR results for REDD1, TP53INP1, p21 and CARF transcripts show increased expression in quiescent fibroblasts relative to proliferating fibroblasts. Y -axis indicates fold enrichment in quiescent versus proliferating fibroblasts. ( E ) qRT-PCR shows miR-22 suppresses TP53INP1, REDD1 and p21 transcript levels in quiescent fibroblasts. Fold changes are denoted on the Y -axis relative to the control siRNA transfection. ( F ) REDD1, TP53INP1, p21 and CARF protein expression in fibroblasts was downregulated by transfection with miR-22 compared with control siRNA transfection. For B, D and E, GAPDH was used as a control for normalizing input RNA levels. For B, C, D and E, bars indicate the mean, and error bars denote ±SD, n = 3. P -values were estimated by Student’s t -test. * P < 0.05.

Article Snippet: Membranes were blocked with 5% milk in TBST and probed with corresponding primary antibodies against specific proteins [high mobility group box-1 (HMGB1): Cell Signaling Technology, IRF5: Abcam ab33478, REDD1: Abcam ab106356, TP53INP1: Abcam ab9755, p21: Abcam ab7960, CARF: ab88322, MXD4: Santa Cruz Biotechnology sc-771, MYC: Santa Cruz Biotechnology sc-764X].

Techniques: Immunoprecipitation, Quantitative RT-PCR, Transfection, Isolation, Control, Luciferase, Plasmid Preparation, Mutagenesis, Expressing

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) Domain architecture of Bacteroidales Cad1. The protein contains a N-terminal CARF domain followed by a linker (L1) and a C-terminal adenosine deaminase (ADA) domain. Residue numbers are labeled. (B) Cad1 used in this study was identified within a type III CRISPR- cas locus in a contig from an unknown Bacteroidales bacterium. (C) Association of Cad1 homologs with different subtypes of type III CRISPR-Cas systems (88 non-redundant homologs used for quantification). (D) Growth of staphylococci carrying pTarget and different pCRISPR variants, measured as OD 600 after the addition of aTc. Mean of three biological triplicates, ±SEM, is reported. (E) Enumeration of colony-forming units (CFUs) from staphylococcal cultures carrying different pCRISPR variants after the addition of aTc. At the indicated times after induction, aliquots were removed and plated on solid medium with or without aTc to count the remaining viable cells. Mean of three biological replicates, ±SEM, is reported. (F) Time course microscopy of S. aureus cells harboring pTarget and pCRISPR(Δspc) or pCRISPR(Cad1) at different times after addition of aTc, experiment repeated for two biological replicates. Scale bar, 4 μM. (G) Quantification of ITP/ATP ratios from bacterial lysates. Extracts from staphylococci harboring pTarget and pCRISPR(Δspc) or pCRISPR(Cad1) were either collected before (0 min) or after (15 min) incubation with aTc and analyzed via LC-MS. Mean of five biological replicates ±SEM, is reported. p values, obtained with a two-sided t test with Welch’s correction, are shown. See also and .

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Residue, Labeling, CRISPR, Microscopy, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) HPLC analysis of cOAs associated with purified Cad1-His 6 . cA 4 and cA 6 standards were used as controls. (B) ITC binding curve of Cad1-CARF-His 6 to cA 4 and cA 6 representing the FitX and FitY values estimated by MicroCal PEAQ-ITC analysis software (Malvern). K d values are ~700 and ~30 nM, respectively. (C) Crystal structure of dimeric apo-Cad1-CARF-His 6 . The predicted ligand binding pocket is shown, and the C-terminal legs from each monomer are colored in cyan. The angle between the C-terminal legs is 58°. (D) cA 6 -bound structure of Cad1-CARF-His 6 showing the ligand at the dimeric interface of the CARF domains, with the angle between the C-terminal legs becoming 55°. (E) cA 4 -bound structure of Cad1-CARF-His 6 showing an increase in the spread of the C-terminal legs to 76°. (F) Growth of staphylococci carrying pTarget and pCRISPR(Cad1) harboring alanine substitutions of Cad1 residues involved in cOA binding, measured as the OD 600 value after 220 min of addition of aTc. Mean of four biological replicates, ±SEM, is reported. See also .

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Purification, Binding Assay, Software, Ligand Binding Assay

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) Cryo-EM structure of hexameric apo-Cad1 illustrating the arrangement of AB, CD, and EF Cad1 dimers (in green, blue, and pink, respectively) aligned in a 3-fold symmetric arrangement (black center triangle). A pocket is formed on the interface of the CARF and ADA domains, and the distance between these domains is 15 Å (closed) on one side of the Cad1 dimer and increases to 24 Å (open) on the opposite side. The pocket is generated by the tilting of the CARF head domain (black dotted arrows). (B) Cryo-EM structure of ATP-bound hexameric Cad1 (conformation 1) displaying AB, CD, and EF dimers in green, blue, and pink, respectively. Insets highlight the residues at one of the inter-domain ATP binding sites (red border, with red arrowheads pointing at the ATP) and one of the empty deaminase pockets (black border). The metal is modeled as Mg +2 . (C) Growth of staphylococci carrying pTarget and pCRISPR(Cad1) harboring the amino acid substitutions of the residues lining the inter-domain ATP binding pocket, measured as the OD 600 value after 220 min of addition of aTc. Mean of three biological triplicates, ±SEM, is reported. (D) Cryo-EM structure of hexameric Cad1 protein in presence of ATP and cA 4 , displaying AB, CD, and EF dimers in green, blue, and pink, respectively. cA 4 , ATP, and ATP/ITP molecules are shown in space-filling representation. Insets highlight the residues at one of the inter-domain ATP binding sites (red border, with red arrowheads pointing at the ATP) and one of the deaminase pockets (black border). The metal is modeled as Mg +2 . See also , , , and .

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Cryo-EM Sample Prep, Generated, Binding Assay

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) Amino acid residues in the deaminase pocket of the apo-Cad1-ATP structure. Dotted lines indicate the distances (in Å) between side chains and the metal ion (modeled as Mg +2 ). (B) Same as (A) but for the deaminase pocket of cA 4 -Cad1-ATP. (C) Superimposition of the structures shown in (A) and (B). The shift in the H448 residue in the ATP-bound cA 4 -Cad1 structure is pointed out by a gray arrow. (D) Superposition of the deaminase pocket of the apo-Cad1-ATP structure (silver) and the cA 4 -Cad1-ATP structure (green). The shift in the loop containing H448 and E451 residues is shown by a red double arrow. The distance between the Cα atoms of E451 in the apo-Cad1-ATP structure (black arrowhead) and in the cA 4 -Cad1-ATP structure (red arrowhead) is marked by black dashed line. (E) Growth of staphylococci carrying pTarget and pCRISPR(Cad1) harboring the amino acid substitutions of the residues involved in ATP/ITP interactions at the deaminase site, measured as the OD 600 value after 220 min of addition of aTc. Mean of four biological replicates, ±SEM, is reported. (F) Cryo-EM structure of hexameric cA 6 -bound Cad1 in the presence of ATP. cA 6 was partly modeled within the CARF binding pocket due to the lack of density. ATP was modeled in three inter-domain binding sites (red border, with red arrowheads pointing at the ATP), and adenine was modeled at the other three sites (gray arrowheads). ATP/ITP and Mg +2 were modeled in four of the six deaminase pockets (black border inset) and only phosphate groups of ATP/ITP and Mg +2 in the other two due to lack of density. See also .

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Residue, Cryo-EM Sample Prep, Binding Assay

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) HPLC analysis of Cad1 (wild-type and mutant versions; 2 μM) reaction products in the presence or absence of ATP (1 mM) and different cOAs (20 μM). Chromatograms of ATP and ITP are shown as standards. Reactions were performed in duplicate. (B) Same as (A) but in the presence of ATP and 100 nM of the indicated cOA. (C) Quantification of the product peaks obtained in (B) as percent of ATP substrate deaminated by Cad1. Reactions performed in triplicate, ±SEM, are reported. p values, obtained with a two-sided t test with Welch’s correction, are shown. (D) HPLC analysis of Cad1 (2 μM) reaction products in the presence adenosine, AMP or ATP (1 mM), and cA 6 (20 μM). Chromatograms of adenosine and AMP are shown as standards. Reactions were performed in duplicate. (E) Same as (D) but using dATP as a substrate. (F) Same as (D) but using CTP as substrate. (G) HPLC chromatograms of Cad1 incubated with the indicated cOA at 500 μM. AMP is provided as a standard. Products of ring nuclease activity would be expected to run between cA 6 and AMP. (H) Quantification of the percent of ATP substrate deaminated by Cad1 incubated with different divalent cations at a concentration of 1 mM except for Zn 2+ , which was incubated at a concentration of 100 μM to mitigate oxidation-induced aggregation of Cad1. A reaction using Mg +2 was performed in the presence of 3 mM EDTA. Reactions were performed in triplicate, and areas under the curve for ATP and ITP peaks were used to determine the % deamination. See also .

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Mutagenesis, Incubation, Activity Assay, Concentration Assay

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) HPLC analysis of reactions products of incubation of different Cad1 fractions (H, hexameric; M, megadalton) in the presence of cA 6 and ATP. Reactions were performed in duplicate. (B) Side view of the AB and CD dimers in the apo-Cad1 hexamer structure illustrating the residues present at the dimer-dimer interface. The residues mutated in this study are shown in the inset. (C) SECMALS analysis of hexameric Cad1 K342A (412 kDa ± 0.485%). (D) SECMALS analysis of hexameric Cad1 E408A (394 kDa ± 0.378%). (E) Growth of staphylococci carrying pTarget and pCRISPR(Cad1) harboring alanine substitutions of residues thought to be involved in the association of Cad1 dimers, measured as the OD 600 value after 220 min of addition of aTc. Mean of four biological replicates, ±SEM, is reported. (F) SECMALS analysis of purified dimeric Cad1 W349A (139 kDa ± 0.518%).

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Incubation, Purification

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Virus, Recombinant, Sequencing, Software

Journal: PLoS ONE

Article Title: Genome-Wide Identification of Calcium-Response Factor (CaRF) Binding Sites Predicts a Role in Regulation of Neuronal Signaling Pathways

doi: 10.1371/journal.pone.0010870

Figure Lengend Snippet: a) WebLogo ( http://weblogo.berkeley.edu/ ) representation of the 10bp motif discovered by the PRIORITY motif finder in the ChIP peak sequences. The height of each letter represents the enrichment of that base at each position. If all four bases are equally represented, no base is shown at that position. b) Competition EMSA analysis of CaRF binding to the consensus chCaRE motif in the ChIP peak of the Camk2n1 gene (camCaRE). Arrow indicates the CaRF-camCaRE complex, and the right triangles indicate increasing concentrations (50 or 100 fold molar excess) of the unlabeled competitor probes. c) Competition EMSA analysis to examine the relative importance of each base across the 10bp chCaRE motif. Recombinant CaRF was incubated with radiolabeled camCaRE in the absence (-) or presence of a 50 or 100-fold molar excess of unlabeled competitor probes. The right triangle indicates increasing competitor concentrations. Competitor probes were based on the camCaRE sequence ( AAAGCGAGGC ) with the indicated changes at each position (e.g. 1G has a G rather than an A at position 1 of the motif while the rest of the motif is unchanged). Degenerate code: Y = C/T, N = A,C,G, or T, B = C,G, or T, R = A/G, H = A, C, or T, D = A, G, or T. d) Alignment of the cCaRE, mCaRE, chCaRE, and camCaRE sequences. The mCaRE, which fails to bind CaRF, differs from the CaRF binding sequences at 5 positions, which are shown in gray. Degenerate bases are as described above along with S = C/G.

Article Snippet: To characterize CaRF binding to the chCaRE sequences, we first tested the ability of CaRF to bind the specific chCaRE motif ( 5′-AAAGCGAGGC-3′ ) found in a CaRF ChIP peak from the promoter of the Camk2n1 gene (camCaRE, ).

Techniques: Binding Assay, Recombinant, Incubation, Sequencing

Journal: PLoS ONE

Article Title: Genome-Wide Identification of Calcium-Response Factor (CaRF) Binding Sites Predicts a Role in Regulation of Neuronal Signaling Pathways

doi: 10.1371/journal.pone.0010870

Figure Lengend Snippet: a) Primary data from the UCSC genome browser ( http://genome.ucsc.edu ) showing the CaRF ChIP peak overlapping exon 1 of the Carf gene. b) Position of CaRF-binding motifs in the CaRF ChIP peak from the Carf gene. Capital letters denote exon 1. The underlined sequences show the two potential CaRF-binding motifs. The more 3′ motif in intron 1 was identified by the PRIORITY motif finder. c) Competition EMSA analysis demonstrates that CaRF can bind both motifs in the Carf ChIP peak. Recombinant CaRF was bound to a radiolabeled cCaRE probe in the absence (-) or presence of a 100-fold molar excess of competitor probes. Arrow indicates the CaRF-cCaRE complex. Unlabeled probes used as competitors are listed across the top. d) Expression of Carf mRNA in a Carf exon 8 KO mouse. Cortical neurons from individual P0 WT or CaRF exon 8 deleted (KO) mice were cultured for 5 days, treated with 1µM TTX overnight, then RNA was harvested for cDNA synthesis and quantitative PCR. Carf mRNA was detected with primers against exons 11–12 distal to the deleted region in Carf . Carf mRNA expression was normalized for expression of Gapdh in the same sample to control for sample handling. e) Chromatin immunoprecipitation for RNA polymerase II on the Carf promoter. Carf promoter DNA co-precipitated with an anti-RNA polymerase II antibody or control IgG was quantitated by Q-PCR, and normalized as a percent of signal in the input DNA. Bars show the mean and error bars show SEM. * p <0.05.

Article Snippet: To characterize CaRF binding to the chCaRE sequences, we first tested the ability of CaRF to bind the specific chCaRE motif ( 5′-AAAGCGAGGC-3′ ) found in a CaRF ChIP peak from the promoter of the Camk2n1 gene (camCaRE, ).

Techniques: Binding Assay, Recombinant, Expressing, Cell Culture, cDNA Synthesis, Real-time Polymerase Chain Reaction, Control, Chromatin Immunoprecipitation