Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) The domain structure of the short (ADAR1p110) and long (ADAR1p150) isoforms of ADAR1 are shown. ADAR1p110 contains a deaminase domain which is responsible for ADAR1’s catalytic deamination activity, three double-stranded RNA binding domains (dsRBDs) which interact with the A-form structure of dsRNA, a Nuclear Localization Sequence (NLS) and a Zβ domain of unknown function. ADAR1p150 contains the same domain structure but has a ∼300 a.a. N-terminal extension which contains a Nuclear Export Sequence (NES) as well as a Zα domain. ( B ) ADAR1 deaminases adenosine to inosine in dsRNA, which replaces the amino group on the adenosine with a keto group and disrupts A-form helical structure at AU base pairs. ( C ) Cartoon model depicting editing of a dsRNA by ADAR1 and the different domains. ( D ) The Zα domain is able to stabilize the left-handed Z-conformation of dsDNA and dsRNA through key residues which stabilize the unique Z-form geometry.

Article Snippet: After blocking, they were incubated with either rabbit anti-ADAR1 (Cell Signaling, #14175) or rabbit anti-ADAR1p150 (Cell Signaling, #32136) primary antibodies at a 1:100 dilution in overnight at 4°C.

Techniques: Activity Assay, RNA Binding Assay, Sequencing

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Domain architectures and point mutant locations for the different re-integrated ADAR1p150 mutant constructs. Red stars indicated mutation sites. ( B ) Western blot (left) and quantification (right) showing doxycycline-inducible expression levels of the re-integrated ADAR1p150 mutants relative to re-integrated wild-type ADAR1p150. Quantification was from three replicates, all of which are shown in Supplemental Figures 5 and 6. ( C ) mRNA expression levels in Transcripts Per Million (TPM, on the y-axis ) of the re-integrated ADAR1p150 constructs for each cell line from RNA-seq data. For each transgene, the detected expression value is indicated for each sample. ( D ) Principal Component Analysis (PCA) of gene expression of the wild-type HEK293T cells, ADAR1 KO and ADAR1p150 KO HEK293T cells, and the ADAR1p150 re-integrated cell lines.

Article Snippet: After blocking, they were incubated with either rabbit anti-ADAR1 (Cell Signaling, #14175) or rabbit anti-ADAR1p150 (Cell Signaling, #32136) primary antibodies at a 1:100 dilution in overnight at 4°C.

Techniques: Mutagenesis, Construct, Western Blot, Expressing, RNA Sequencing, Gene Expression

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Immunofluorescence images of the wild-type, ADAR1 KO, ADAR1p150 KO, re-integrated ADAR1p150 (ADAR1p150*), the N173S mutant (ADAR1p150 N173S *), and the N173A,Y177A (ADAR1p150 N173A,Y177A *) double mutant cell lines. The red signal is of an ADAR1p150-specific rabbit monoclonal antibody visualized using an Alexa Fluor 594 nm secondary antibody. The green signal is from G3BP1 which was endogenously tagged with EGFP, and the blue signal is DAPI. ( B ) The percentage of the red signal intensity (corresponding to the anti-ADAR1p150 antibody) in the cytoplasm versus the nucleus of the cell.

Article Snippet: After blocking, they were incubated with either rabbit anti-ADAR1 (Cell Signaling, #14175) or rabbit anti-ADAR1p150 (Cell Signaling, #32136) primary antibodies at a 1:100 dilution in overnight at 4°C.

Techniques: Immunofluorescence, Mutagenesis

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) The structure of the Zα domain of ADAR1 bound to Z-RNA is shown (PDB: 2GXB) highlighting the water-mediated hydrogen bond between N173 and W195. ( B ) Domain structures of the ADAR1p150 N173S and ADAR1p150 N173S NES moved constructs. ( C ) Immunofluorescence images of the re-integrated wild-type ADAR1p150*, ADAR1p150 N173S , and ADAR1p150 N173S * NES moved cell lines. ( D ) percentage of Alexa Fluor 594 signal intensity (corresponding to anti-ADAR1p150 antibody staining) measured in the cytoplasm versus the nucleus of the cell for the ADAR1p150 N173S and ADAR1p150 N173S * NES moved cell lines.

Article Snippet: After blocking, they were incubated with either rabbit anti-ADAR1 (Cell Signaling, #14175) or rabbit anti-ADAR1p150 (Cell Signaling, #32136) primary antibodies at a 1:100 dilution in overnight at 4°C.

Techniques: Construct, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Domain structures of ADAR1p150 and ZBP1 are shown. ( B ) A cartoon depiction of the two potential models showing the effect of the Zα domain on ADAR1 function. In model 1, the Zα domain augments A-to-I editing broadly in a sequence-independent manner. In model 2, the Zα domain of ADAR1 competes with ZBP1 for binding to Z-form substrates in an editing independent manner, thereby inhibiting cell death pathways.

Article Snippet: After blocking, they were incubated with either rabbit anti-ADAR1 (Cell Signaling, #14175) or rabbit anti-ADAR1p150 (Cell Signaling, #32136) primary antibodies at a 1:100 dilution in overnight at 4°C.

Techniques: Sequencing, Binding Assay

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) The domain structure of the short (ADAR1p110) and long (ADAR1p150) isoforms of ADAR1 are shown. ADAR1p110 contains a deaminase domain which is responsible for ADAR1’s catalytic deamination activity, three double-stranded RNA binding domains (dsRBDs) which interact with the A-form structure of dsRNA, a Nuclear Localization Sequence (NLS) and a Zβ domain of unknown function. ADAR1p150 contains the same domain structure but has a ∼300 a.a. N-terminal extension which contains a Nuclear Export Sequence (NES) as well as a Zα domain. ( B ) ADAR1 deaminases adenosine to inosine in dsRNA, which replaces the amino group on the adenosine with a keto group and disrupts A-form helical structure at AU base pairs. ( C ) Cartoon model depicting editing of a dsRNA by ADAR1 and the different domains. ( D ) The Zα domain is able to stabilize the left-handed Z-conformation of dsDNA and dsRNA through key residues which stabilize the unique Z-form geometry.

Article Snippet: Western blots of ADAR1p150 were carried out identically as above using a rabbit anti-ADAR1p150 antibody (Cell Signaling, #32136).

Techniques: Activity Assay, RNA Binding Assay, Sequencing

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Domain architectures and point mutant locations for the different re-integrated ADAR1p150 mutant constructs. Red stars indicated mutation sites. ( B ) Western blot (left) and quantification (right) showing doxycycline-inducible expression levels of the re-integrated ADAR1p150 mutants relative to re-integrated wild-type ADAR1p150. Quantification was from three replicates, all of which are shown in Supplemental Figures 5 and 6. ( C ) mRNA expression levels in Transcripts Per Million (TPM, on the y-axis ) of the re-integrated ADAR1p150 constructs for each cell line from RNA-seq data. For each transgene, the detected expression value is indicated for each sample. ( D ) Principal Component Analysis (PCA) of gene expression of the wild-type HEK293T cells, ADAR1 KO and ADAR1p150 KO HEK293T cells, and the ADAR1p150 re-integrated cell lines.

Article Snippet: Western blots of ADAR1p150 were carried out identically as above using a rabbit anti-ADAR1p150 antibody (Cell Signaling, #32136).

Techniques: Mutagenesis, Construct, Western Blot, Expressing, RNA Sequencing, Gene Expression

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Illustration of how A-to-I editing events from aligned reads and unaligned reads are identified. Editing indices (the ratio of total A-to-G changes over the total number of adenosines) calculated for all REDIprotal sites ( B ) and Alu elements specifically ( C , AEI = Alu Editing Index) are shown for the different cell lines. Comparisons referenced to in the main text are marked and numbered. Each black dot represents a replicate and the red cross is the average of three replicates. ( D ) Similar to the editing index, the number of hyper-edited sites per million reads from originally unmapped reads is shown. ( E ) The editing index for ADAR1p150-specific REDIportal sites (by filtering out sites observed in the ADAR1p150 KO cell line) for all cell lines is shown. ( F ) The numbers of differentially edited sites for individual cell line pairs are compared y-axis as determined using DESeq2 . ( G ) Editing frequencies at the top 3 most differentially edited sites between wild-type and ADAR1p150 KO are shown.

Article Snippet: Western blots of ADAR1p150 were carried out identically as above using a rabbit anti-ADAR1p150 antibody (Cell Signaling, #32136).

Techniques:

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Illustration of how editing clusters are identified, figure adapted from . ( B ) The cluster editing index, calculated by taking the number of A-to-G changes over the total number of adenosines per identified cluster, then averaged for all clusters. Comparisons which are mentioned in the main text are marked and numbered. Each black dot represents a replicate and the red cross is the average of three replicates. ( C ) The number of differentially edited clusters for individual cell line pairs are compared on the y-axis as determined using DESeq2 . ( D ) Zoomed in fragments of Alu foldbacks predicted by the CRSSANT software package that were also ADAR1p150-dependent (ie, they have differential editing when comparing the ADAR1p150* plus and minus doxycycline conditions). Editing sites were only plotted on the predicted dsRNAs if they had an average editing frequency of > 0.05 averaged over the 3 replicates.

Article Snippet: Western blots of ADAR1p150 were carried out identically as above using a rabbit anti-ADAR1p150 antibody (Cell Signaling, #32136).

Techniques: Software

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Immunofluorescence images of the wild-type, ADAR1 KO, ADAR1p150 KO, re-integrated ADAR1p150 (ADAR1p150*), the N173S mutant (ADAR1p150 N173S *), and the N173A,Y177A (ADAR1p150 N173A,Y177A *) double mutant cell lines. The red signal is of an ADAR1p150-specific rabbit monoclonal antibody visualized using an Alexa Fluor 594 nm secondary antibody. The green signal is from G3BP1 which was endogenously tagged with EGFP, and the blue signal is DAPI. ( B ) The percentage of the red signal intensity (corresponding to the anti-ADAR1p150 antibody) in the cytoplasm versus the nucleus of the cell.

Article Snippet: Western blots of ADAR1p150 were carried out identically as above using a rabbit anti-ADAR1p150 antibody (Cell Signaling, #32136).

Techniques: Immunofluorescence, Mutagenesis

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) The structure of the Zα domain of ADAR1 bound to Z-RNA is shown (PDB: 2GXB) highlighting the water-mediated hydrogen bond between N173 and W195. ( B ) Domain structures of the ADAR1p150 N173S and ADAR1p150 N173S NES moved constructs. ( C ) Immunofluorescence images of the re-integrated wild-type ADAR1p150*, ADAR1p150 N173S , and ADAR1p150 N173S * NES moved cell lines. ( D ) percentage of Alexa Fluor 594 signal intensity (corresponding to anti-ADAR1p150 antibody staining) measured in the cytoplasm versus the nucleus of the cell for the ADAR1p150 N173S and ADAR1p150 N173S * NES moved cell lines.

Article Snippet: Western blots of ADAR1p150 were carried out identically as above using a rabbit anti-ADAR1p150 antibody (Cell Signaling, #32136).

Techniques: Construct, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Domain structures of ADAR1p150 and ZBP1 are shown. ( B ) A cartoon depiction of the two potential models showing the effect of the Zα domain on ADAR1 function. In model 1, the Zα domain augments A-to-I editing broadly in a sequence-independent manner. In model 2, the Zα domain of ADAR1 competes with ZBP1 for binding to Z-form substrates in an editing independent manner, thereby inhibiting cell death pathways.

Article Snippet: Western blots of ADAR1p150 were carried out identically as above using a rabbit anti-ADAR1p150 antibody (Cell Signaling, #32136).

Techniques: Sequencing, Binding Assay

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) The domain structure of the short (ADAR1p110) and long (ADAR1p150) isoforms of ADAR1 are shown. ADAR1p110 contains a deaminase domain which is responsible for ADAR1’s catalytic deamination activity, three double-stranded RNA binding domains (dsRBDs) which interact with the A-form structure of dsRNA, a Nuclear Localization Sequence (NLS) and a Zβ domain of unknown function. ADAR1p150 contains the same domain structure but has a ∼300 a.a. N-terminal extension which contains a Nuclear Export Sequence (NES) as well as a Zα domain. ( B ) ADAR1 deaminases adenosine to inosine in dsRNA, which replaces the amino group on the adenosine with a keto group and disrupts A-form helical structure at AU base pairs. ( C ) Cartoon model depicting editing of a dsRNA by ADAR1 and the different domains. ( D ) The Zα domain is able to stabilize the left-handed Z-conformation of dsDNA and dsRNA through key residues which stabilize the unique Z-form geometry.

Article Snippet: After blocking, they were incubated with either rabbit anti-ADAR1 (Cell Signaling, #14175) or rabbit anti-ADAR1p150 (Cell Signaling, #32136) primary antibodies at a 1:100 dilution in overnight at 4°C.

Techniques: Activity Assay, RNA Binding Assay, Sequencing

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Domain architectures and point mutant locations for the different re-integrated ADAR1p150 mutant constructs. Red stars indicated mutation sites. ( B ) Western blot (left) and quantification (right) showing doxycycline-inducible expression levels of the re-integrated ADAR1p150 mutants relative to re-integrated wild-type ADAR1p150. Quantification was from three replicates, all of which are shown in Supplemental Figures 5 and 6. ( C ) mRNA expression levels in Transcripts Per Million (TPM, on the y-axis ) of the re-integrated ADAR1p150 constructs for each cell line from RNA-seq data. For each transgene, the detected expression value is indicated for each sample. ( D ) Principal Component Analysis (PCA) of gene expression of the wild-type HEK293T cells, ADAR1 KO and ADAR1p150 KO HEK293T cells, and the ADAR1p150 re-integrated cell lines.

Article Snippet: After blocking, they were incubated with either rabbit anti-ADAR1 (Cell Signaling, #14175) or rabbit anti-ADAR1p150 (Cell Signaling, #32136) primary antibodies at a 1:100 dilution in overnight at 4°C.

Techniques: Mutagenesis, Construct, Western Blot, Expressing, RNA Sequencing, Gene Expression

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Illustration of how A-to-I editing events from aligned reads and unaligned reads are identified. Editing indices (the ratio of total A-to-G changes over the total number of adenosines) calculated for all REDIprotal sites ( B ) and Alu elements specifically ( C , AEI = Alu Editing Index) are shown for the different cell lines. Comparisons referenced to in the main text are marked and numbered. Each black dot represents a replicate and the red cross is the average of three replicates. ( D ) Similar to the editing index, the number of hyper-edited sites per million reads from originally unmapped reads is shown. ( E ) The editing index for ADAR1p150-specific REDIportal sites (by filtering out sites observed in the ADAR1p150 KO cell line) for all cell lines is shown. ( F ) The numbers of differentially edited sites for individual cell line pairs are compared y-axis as determined using DESeq2 . ( G ) Editing frequencies at the top 3 most differentially edited sites between wild-type and ADAR1p150 KO are shown.

Article Snippet: After blocking, they were incubated with either rabbit anti-ADAR1 (Cell Signaling, #14175) or rabbit anti-ADAR1p150 (Cell Signaling, #32136) primary antibodies at a 1:100 dilution in overnight at 4°C.

Techniques:

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Illustration of how editing clusters are identified, figure adapted from . ( B ) The cluster editing index, calculated by taking the number of A-to-G changes over the total number of adenosines per identified cluster, then averaged for all clusters. Comparisons which are mentioned in the main text are marked and numbered. Each black dot represents a replicate and the red cross is the average of three replicates. ( C ) The number of differentially edited clusters for individual cell line pairs are compared on the y-axis as determined using DESeq2 . ( D ) Zoomed in fragments of Alu foldbacks predicted by the CRSSANT software package that were also ADAR1p150-dependent (ie, they have differential editing when comparing the ADAR1p150* plus and minus doxycycline conditions). Editing sites were only plotted on the predicted dsRNAs if they had an average editing frequency of > 0.05 averaged over the 3 replicates.

Article Snippet: After blocking, they were incubated with either rabbit anti-ADAR1 (Cell Signaling, #14175) or rabbit anti-ADAR1p150 (Cell Signaling, #32136) primary antibodies at a 1:100 dilution in overnight at 4°C.

Techniques: Software

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Immunofluorescence images of the wild-type, ADAR1 KO, ADAR1p150 KO, re-integrated ADAR1p150 (ADAR1p150*), the N173S mutant (ADAR1p150 N173S *), and the N173A,Y177A (ADAR1p150 N173A,Y177A *) double mutant cell lines. The red signal is of an ADAR1p150-specific rabbit monoclonal antibody visualized using an Alexa Fluor 594 nm secondary antibody. The green signal is from G3BP1 which was endogenously tagged with EGFP, and the blue signal is DAPI. ( B ) The percentage of the red signal intensity (corresponding to the anti-ADAR1p150 antibody) in the cytoplasm versus the nucleus of the cell.

Article Snippet: After blocking, they were incubated with either rabbit anti-ADAR1 (Cell Signaling, #14175) or rabbit anti-ADAR1p150 (Cell Signaling, #32136) primary antibodies at a 1:100 dilution in overnight at 4°C.

Techniques: Immunofluorescence, Mutagenesis

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) The structure of the Zα domain of ADAR1 bound to Z-RNA is shown (PDB: 2GXB) highlighting the water-mediated hydrogen bond between N173 and W195. ( B ) Domain structures of the ADAR1p150 N173S and ADAR1p150 N173S NES moved constructs. ( C ) Immunofluorescence images of the re-integrated wild-type ADAR1p150*, ADAR1p150 N173S , and ADAR1p150 N173S * NES moved cell lines. ( D ) percentage of Alexa Fluor 594 signal intensity (corresponding to anti-ADAR1p150 antibody staining) measured in the cytoplasm versus the nucleus of the cell for the ADAR1p150 N173S and ADAR1p150 N173S * NES moved cell lines.

Article Snippet: After blocking, they were incubated with either rabbit anti-ADAR1 (Cell Signaling, #14175) or rabbit anti-ADAR1p150 (Cell Signaling, #32136) primary antibodies at a 1:100 dilution in overnight at 4°C.

Techniques: Construct, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Domain structures of ADAR1p150 and ZBP1 are shown. ( B ) A cartoon depiction of the two potential models showing the effect of the Zα domain on ADAR1 function. In model 1, the Zα domain augments A-to-I editing broadly in a sequence-independent manner. In model 2, the Zα domain of ADAR1 competes with ZBP1 for binding to Z-form substrates in an editing independent manner, thereby inhibiting cell death pathways.

Article Snippet: After blocking, they were incubated with either rabbit anti-ADAR1 (Cell Signaling, #14175) or rabbit anti-ADAR1p150 (Cell Signaling, #32136) primary antibodies at a 1:100 dilution in overnight at 4°C.

Techniques: Sequencing, Binding Assay

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) The domain structure of the short (ADAR1p110) and long (ADAR1p150) isoforms of ADAR1 are shown. ADAR1p110 contains a deaminase domain which is responsible for ADAR1’s catalytic deamination activity, three double-stranded RNA binding domains (dsRBDs) which interact with the A-form structure of dsRNA, a Nuclear Localization Sequence (NLS) and a Zβ domain of unknown function. ADAR1p150 contains the same domain structure but has a ∼300 a.a. N-terminal extension which contains a Nuclear Export Sequence (NES) as well as a Zα domain. ( B ) ADAR1 deaminases adenosine to inosine in dsRNA, which replaces the amino group on the adenosine with a keto group and disrupts A-form helical structure at AU base pairs. ( C ) Cartoon model depicting editing of a dsRNA by ADAR1 and the different domains. ( D ) The Zα domain is able to stabilize the left-handed Z-conformation of dsDNA and dsRNA through key residues which stabilize the unique Z-form geometry.

Article Snippet: After blocking, the membrane was incubated overnight at 4°C with a rabbit anti-ADAR1 primary antibody (Cell Signaling, #14175) diluted 1:1000 in blocking buffer.

Techniques: Activity Assay, RNA Binding Assay, Sequencing

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Domain architectures and point mutant locations for the different re-integrated ADAR1p150 mutant constructs. Red stars indicated mutation sites. ( B ) Western blot (left) and quantification (right) showing doxycycline-inducible expression levels of the re-integrated ADAR1p150 mutants relative to re-integrated wild-type ADAR1p150. Quantification was from three replicates, all of which are shown in Supplemental Figures 5 and 6. ( C ) mRNA expression levels in Transcripts Per Million (TPM, on the y-axis ) of the re-integrated ADAR1p150 constructs for each cell line from RNA-seq data. For each transgene, the detected expression value is indicated for each sample. ( D ) Principal Component Analysis (PCA) of gene expression of the wild-type HEK293T cells, ADAR1 KO and ADAR1p150 KO HEK293T cells, and the ADAR1p150 re-integrated cell lines.

Article Snippet: After blocking, the membrane was incubated overnight at 4°C with a rabbit anti-ADAR1 primary antibody (Cell Signaling, #14175) diluted 1:1000 in blocking buffer.

Techniques: Mutagenesis, Construct, Western Blot, Expressing, RNA Sequencing, Gene Expression

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Immunofluorescence images of the wild-type, ADAR1 KO, ADAR1p150 KO, re-integrated ADAR1p150 (ADAR1p150*), the N173S mutant (ADAR1p150 N173S *), and the N173A,Y177A (ADAR1p150 N173A,Y177A *) double mutant cell lines. The red signal is of an ADAR1p150-specific rabbit monoclonal antibody visualized using an Alexa Fluor 594 nm secondary antibody. The green signal is from G3BP1 which was endogenously tagged with EGFP, and the blue signal is DAPI. ( B ) The percentage of the red signal intensity (corresponding to the anti-ADAR1p150 antibody) in the cytoplasm versus the nucleus of the cell.

Article Snippet: After blocking, the membrane was incubated overnight at 4°C with a rabbit anti-ADAR1 primary antibody (Cell Signaling, #14175) diluted 1:1000 in blocking buffer.

Techniques: Immunofluorescence, Mutagenesis

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) The structure of the Zα domain of ADAR1 bound to Z-RNA is shown (PDB: 2GXB) highlighting the water-mediated hydrogen bond between N173 and W195. ( B ) Domain structures of the ADAR1p150 N173S and ADAR1p150 N173S NES moved constructs. ( C ) Immunofluorescence images of the re-integrated wild-type ADAR1p150*, ADAR1p150 N173S , and ADAR1p150 N173S * NES moved cell lines. ( D ) percentage of Alexa Fluor 594 signal intensity (corresponding to anti-ADAR1p150 antibody staining) measured in the cytoplasm versus the nucleus of the cell for the ADAR1p150 N173S and ADAR1p150 N173S * NES moved cell lines.

Article Snippet: After blocking, the membrane was incubated overnight at 4°C with a rabbit anti-ADAR1 primary antibody (Cell Signaling, #14175) diluted 1:1000 in blocking buffer.

Techniques: Construct, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Domain structures of ADAR1p150 and ZBP1 are shown. ( B ) A cartoon depiction of the two potential models showing the effect of the Zα domain on ADAR1 function. In model 1, the Zα domain augments A-to-I editing broadly in a sequence-independent manner. In model 2, the Zα domain of ADAR1 competes with ZBP1 for binding to Z-form substrates in an editing independent manner, thereby inhibiting cell death pathways.

Article Snippet: After blocking, the membrane was incubated overnight at 4°C with a rabbit anti-ADAR1 primary antibody (Cell Signaling, #14175) diluted 1:1000 in blocking buffer.

Techniques: Sequencing, Binding Assay