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Genomic analysis of ADAR1 binding and its involvement in multiple RNA processing pathways.

Bahn JH, Ahn J, Lin X, Zhang Q, Lee JH, Civelek M, Xiao X - Nat Commun (2015)

Bottom Line: Similarly, ADAR1 interacts with DROSHA and DGCR8 in the nucleus and possibly out-competes DGCR8 in primary miRNA binding, which enhances mature miRNA expression.These functions are dependent on ADAR1's editing activity, at least for a subset of targets.Our study unfolds a broad landscape of the functional roles of ADAR1.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Physiology and the Molecular Biology Institute, University of California Los Angeles, Los Angeles, California 90095, USA.

ABSTRACT
Adenosine deaminases acting on RNA (ADARs) are the primary factors underlying adenosine to inosine (A-to-I) editing in metazoans. Here we report the first global study of ADAR1-RNA interaction in human cells using CLIP-seq. A large number of CLIP sites are observed in Alu repeats, consistent with ADAR1's function in RNA editing. Surprisingly, thousands of other CLIP sites are located in non-Alu regions, revealing functional and biophysical targets of ADAR1 in the regulation of alternative 3' UTR usage and miRNA biogenesis. We observe that binding of ADAR1 to 3' UTRs precludes binding by other factors, causing 3' UTR lengthening. Similarly, ADAR1 interacts with DROSHA and DGCR8 in the nucleus and possibly out-competes DGCR8 in primary miRNA binding, which enhances mature miRNA expression. These functions are dependent on ADAR1's editing activity, at least for a subset of targets. Our study unfolds a broad landscape of the functional roles of ADAR1.

No MeSH data available.


ADAR1 mediates pri-miRNA processing(a) CLIP reads mapped to the mature, precursor and primary transcripts of miR-21-5p. Light and dark gray bars represent the relative locations of annotated mature and pre-miR-21. Stem-loop structure is shown for illustration purpose only that does not reflect the true structure of pre-miR-21. (b) Numbers of mature, pre-miR and pri-miR bound by ADAR1 and numbers of miRNAs with two or three forms bound by ADAR1 (shown as overlaps). Overlap p values between pri-miR and pre-miR: 0.024, between pri-miR and mature: 0.79 and between pre-miR and mature: 0.18, calculated using hypergeometric test and assuming a total of 410 miRNAs being expressed in U87MG cells (based on small RNA sequencing data). (c) Pri- (left panel) and mature miRNA expression (right panel) of endogenous miR-21-5p, miR-34a-5p, and miR-100-5p in U87MG cells as measured by RT-qPCR. Cells were transfected with the pEGFP-C1 vector (GFP), pEGFP-C1-ADAR1 vector (ADAR1), scrambled siRNA (siCtrl) or siRNA for ADAR1 (siADAR1) respectively. Results (mean and SD) from ≥4 biological replicates are shown. #p < 0.01, *p < 0.05 (Wilcoxon Rank-Sum test). (d) Expression change of miRNAs in U87MG cells with ADAR1 perturbation (KD or OE) relative to controls. Only miRNAs whose primary transcripts were associated with ADAR1 CLIP reads are included. Filled circles represent miRNAs with significantly altered expression in KD or OE. If the significance was observed in both experiments or if no significance was found in either experiment, the average expression change is shown. If only one experiment led to a significant change, the value of expression change in this experiment is shown. Two miRNAs were excluded because they demonstrated significant changes in both experiments, but in conflicting directions. (e) Cumulative distribution functions of log2-fold changes (LFCs) of miRNAs bound by ADAR1 in the primary form in U87MG cells upon OE of wildtype ADAR1, E912A or EAA mutants, compared to controls. The LFC values represent log2(OE/ctrls) which were further normalized by spike-in controls to account for technical variations across experiments. (f) ADAR1 associates with both DROSHA and DGCR8. Co-immunoprecipitation (Co-IP) experiments were performed using DROSHA antibody (ab12286), DGCR8 antibody (ab90579), ADAR1 antibody (D-8), or corresponding rabbit (r) or mouse (m) isotype IgG in HeLa cells. HeLa cells were used since DROSHA and DGCR8 expression is relatively higher in HeLa than in U87MG cells. IP samples were immunoblotted (IB) using ADAR1 antibody (15.8.6), DROSHA antibody (ab12286) and DGCR8 antibody (ab90579) to detect the corresponding antigens. ADAR1 interacts with both DROSHA and DGCR8, reciprocally but not the corresponding IgG isotype control. RNase A treatment does not significantly impair the interactions.
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Figure 4: ADAR1 mediates pri-miRNA processing(a) CLIP reads mapped to the mature, precursor and primary transcripts of miR-21-5p. Light and dark gray bars represent the relative locations of annotated mature and pre-miR-21. Stem-loop structure is shown for illustration purpose only that does not reflect the true structure of pre-miR-21. (b) Numbers of mature, pre-miR and pri-miR bound by ADAR1 and numbers of miRNAs with two or three forms bound by ADAR1 (shown as overlaps). Overlap p values between pri-miR and pre-miR: 0.024, between pri-miR and mature: 0.79 and between pre-miR and mature: 0.18, calculated using hypergeometric test and assuming a total of 410 miRNAs being expressed in U87MG cells (based on small RNA sequencing data). (c) Pri- (left panel) and mature miRNA expression (right panel) of endogenous miR-21-5p, miR-34a-5p, and miR-100-5p in U87MG cells as measured by RT-qPCR. Cells were transfected with the pEGFP-C1 vector (GFP), pEGFP-C1-ADAR1 vector (ADAR1), scrambled siRNA (siCtrl) or siRNA for ADAR1 (siADAR1) respectively. Results (mean and SD) from ≥4 biological replicates are shown. #p < 0.01, *p < 0.05 (Wilcoxon Rank-Sum test). (d) Expression change of miRNAs in U87MG cells with ADAR1 perturbation (KD or OE) relative to controls. Only miRNAs whose primary transcripts were associated with ADAR1 CLIP reads are included. Filled circles represent miRNAs with significantly altered expression in KD or OE. If the significance was observed in both experiments or if no significance was found in either experiment, the average expression change is shown. If only one experiment led to a significant change, the value of expression change in this experiment is shown. Two miRNAs were excluded because they demonstrated significant changes in both experiments, but in conflicting directions. (e) Cumulative distribution functions of log2-fold changes (LFCs) of miRNAs bound by ADAR1 in the primary form in U87MG cells upon OE of wildtype ADAR1, E912A or EAA mutants, compared to controls. The LFC values represent log2(OE/ctrls) which were further normalized by spike-in controls to account for technical variations across experiments. (f) ADAR1 associates with both DROSHA and DGCR8. Co-immunoprecipitation (Co-IP) experiments were performed using DROSHA antibody (ab12286), DGCR8 antibody (ab90579), ADAR1 antibody (D-8), or corresponding rabbit (r) or mouse (m) isotype IgG in HeLa cells. HeLa cells were used since DROSHA and DGCR8 expression is relatively higher in HeLa than in U87MG cells. IP samples were immunoblotted (IB) using ADAR1 antibody (15.8.6), DROSHA antibody (ab12286) and DGCR8 antibody (ab90579) to detect the corresponding antigens. ADAR1 interacts with both DROSHA and DGCR8, reciprocally but not the corresponding IgG isotype control. RNase A treatment does not significantly impair the interactions.

Mentions: In addition to coding genes, ADAR1 also interacts with non-coding RNAs within non-Alu regions, particularly miRNA transcripts, most of which do not overlap with Alu repeats. Our CLIP data allowed a genome-wide analysis of the interactions between ADAR1 and miRNA transcripts. We observed that ADAR1 could bind to all three forms of miRNAs: primary (pri-), precursor (pre-), and mature miRNAs (Methods), an example of which is shown in Fig. 4a. Overall, 220, 37, and 25 pri-, pre-, and mature miRNAs were associated with ADAR1, respectively (Fig. 4b & Supplementary Table 6). Among the 3 forms of miRNAs, pri-miRNAs were most often observed with ADAR1 binding, possibly due to their longer length and/or the relative abundance of ADAR1 in the nucleus of U87MG cells (Supplementary Fig. 11). A few miRNAs previously reported to be edited by ADAR1 38, 39 were present in the ADAR1-CLIP primary miRNA list (Supplementary Table 6), supporting our observed interactions between ADAR1 and primary miRNAs. Interestingly, 25 miRNAs were associated with ADAR1 in both precursor and primary transcripts, which is a significant overlap (p = 0.02, hypergeometric test) (Fig. 4b). These data together prompted the hypothesis that ADAR1 may affect pri-miRNA processing through interaction with the primary transcripts.


Genomic analysis of ADAR1 binding and its involvement in multiple RNA processing pathways.

Bahn JH, Ahn J, Lin X, Zhang Q, Lee JH, Civelek M, Xiao X - Nat Commun (2015)

ADAR1 mediates pri-miRNA processing(a) CLIP reads mapped to the mature, precursor and primary transcripts of miR-21-5p. Light and dark gray bars represent the relative locations of annotated mature and pre-miR-21. Stem-loop structure is shown for illustration purpose only that does not reflect the true structure of pre-miR-21. (b) Numbers of mature, pre-miR and pri-miR bound by ADAR1 and numbers of miRNAs with two or three forms bound by ADAR1 (shown as overlaps). Overlap p values between pri-miR and pre-miR: 0.024, between pri-miR and mature: 0.79 and between pre-miR and mature: 0.18, calculated using hypergeometric test and assuming a total of 410 miRNAs being expressed in U87MG cells (based on small RNA sequencing data). (c) Pri- (left panel) and mature miRNA expression (right panel) of endogenous miR-21-5p, miR-34a-5p, and miR-100-5p in U87MG cells as measured by RT-qPCR. Cells were transfected with the pEGFP-C1 vector (GFP), pEGFP-C1-ADAR1 vector (ADAR1), scrambled siRNA (siCtrl) or siRNA for ADAR1 (siADAR1) respectively. Results (mean and SD) from ≥4 biological replicates are shown. #p < 0.01, *p < 0.05 (Wilcoxon Rank-Sum test). (d) Expression change of miRNAs in U87MG cells with ADAR1 perturbation (KD or OE) relative to controls. Only miRNAs whose primary transcripts were associated with ADAR1 CLIP reads are included. Filled circles represent miRNAs with significantly altered expression in KD or OE. If the significance was observed in both experiments or if no significance was found in either experiment, the average expression change is shown. If only one experiment led to a significant change, the value of expression change in this experiment is shown. Two miRNAs were excluded because they demonstrated significant changes in both experiments, but in conflicting directions. (e) Cumulative distribution functions of log2-fold changes (LFCs) of miRNAs bound by ADAR1 in the primary form in U87MG cells upon OE of wildtype ADAR1, E912A or EAA mutants, compared to controls. The LFC values represent log2(OE/ctrls) which were further normalized by spike-in controls to account for technical variations across experiments. (f) ADAR1 associates with both DROSHA and DGCR8. Co-immunoprecipitation (Co-IP) experiments were performed using DROSHA antibody (ab12286), DGCR8 antibody (ab90579), ADAR1 antibody (D-8), or corresponding rabbit (r) or mouse (m) isotype IgG in HeLa cells. HeLa cells were used since DROSHA and DGCR8 expression is relatively higher in HeLa than in U87MG cells. IP samples were immunoblotted (IB) using ADAR1 antibody (15.8.6), DROSHA antibody (ab12286) and DGCR8 antibody (ab90579) to detect the corresponding antigens. ADAR1 interacts with both DROSHA and DGCR8, reciprocally but not the corresponding IgG isotype control. RNase A treatment does not significantly impair the interactions.
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Related In: Results  -  Collection

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Figure 4: ADAR1 mediates pri-miRNA processing(a) CLIP reads mapped to the mature, precursor and primary transcripts of miR-21-5p. Light and dark gray bars represent the relative locations of annotated mature and pre-miR-21. Stem-loop structure is shown for illustration purpose only that does not reflect the true structure of pre-miR-21. (b) Numbers of mature, pre-miR and pri-miR bound by ADAR1 and numbers of miRNAs with two or three forms bound by ADAR1 (shown as overlaps). Overlap p values between pri-miR and pre-miR: 0.024, between pri-miR and mature: 0.79 and between pre-miR and mature: 0.18, calculated using hypergeometric test and assuming a total of 410 miRNAs being expressed in U87MG cells (based on small RNA sequencing data). (c) Pri- (left panel) and mature miRNA expression (right panel) of endogenous miR-21-5p, miR-34a-5p, and miR-100-5p in U87MG cells as measured by RT-qPCR. Cells were transfected with the pEGFP-C1 vector (GFP), pEGFP-C1-ADAR1 vector (ADAR1), scrambled siRNA (siCtrl) or siRNA for ADAR1 (siADAR1) respectively. Results (mean and SD) from ≥4 biological replicates are shown. #p < 0.01, *p < 0.05 (Wilcoxon Rank-Sum test). (d) Expression change of miRNAs in U87MG cells with ADAR1 perturbation (KD or OE) relative to controls. Only miRNAs whose primary transcripts were associated with ADAR1 CLIP reads are included. Filled circles represent miRNAs with significantly altered expression in KD or OE. If the significance was observed in both experiments or if no significance was found in either experiment, the average expression change is shown. If only one experiment led to a significant change, the value of expression change in this experiment is shown. Two miRNAs were excluded because they demonstrated significant changes in both experiments, but in conflicting directions. (e) Cumulative distribution functions of log2-fold changes (LFCs) of miRNAs bound by ADAR1 in the primary form in U87MG cells upon OE of wildtype ADAR1, E912A or EAA mutants, compared to controls. The LFC values represent log2(OE/ctrls) which were further normalized by spike-in controls to account for technical variations across experiments. (f) ADAR1 associates with both DROSHA and DGCR8. Co-immunoprecipitation (Co-IP) experiments were performed using DROSHA antibody (ab12286), DGCR8 antibody (ab90579), ADAR1 antibody (D-8), or corresponding rabbit (r) or mouse (m) isotype IgG in HeLa cells. HeLa cells were used since DROSHA and DGCR8 expression is relatively higher in HeLa than in U87MG cells. IP samples were immunoblotted (IB) using ADAR1 antibody (15.8.6), DROSHA antibody (ab12286) and DGCR8 antibody (ab90579) to detect the corresponding antigens. ADAR1 interacts with both DROSHA and DGCR8, reciprocally but not the corresponding IgG isotype control. RNase A treatment does not significantly impair the interactions.
Mentions: In addition to coding genes, ADAR1 also interacts with non-coding RNAs within non-Alu regions, particularly miRNA transcripts, most of which do not overlap with Alu repeats. Our CLIP data allowed a genome-wide analysis of the interactions between ADAR1 and miRNA transcripts. We observed that ADAR1 could bind to all three forms of miRNAs: primary (pri-), precursor (pre-), and mature miRNAs (Methods), an example of which is shown in Fig. 4a. Overall, 220, 37, and 25 pri-, pre-, and mature miRNAs were associated with ADAR1, respectively (Fig. 4b & Supplementary Table 6). Among the 3 forms of miRNAs, pri-miRNAs were most often observed with ADAR1 binding, possibly due to their longer length and/or the relative abundance of ADAR1 in the nucleus of U87MG cells (Supplementary Fig. 11). A few miRNAs previously reported to be edited by ADAR1 38, 39 were present in the ADAR1-CLIP primary miRNA list (Supplementary Table 6), supporting our observed interactions between ADAR1 and primary miRNAs. Interestingly, 25 miRNAs were associated with ADAR1 in both precursor and primary transcripts, which is a significant overlap (p = 0.02, hypergeometric test) (Fig. 4b). These data together prompted the hypothesis that ADAR1 may affect pri-miRNA processing through interaction with the primary transcripts.

Bottom Line: Similarly, ADAR1 interacts with DROSHA and DGCR8 in the nucleus and possibly out-competes DGCR8 in primary miRNA binding, which enhances mature miRNA expression.These functions are dependent on ADAR1's editing activity, at least for a subset of targets.Our study unfolds a broad landscape of the functional roles of ADAR1.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Physiology and the Molecular Biology Institute, University of California Los Angeles, Los Angeles, California 90095, USA.

ABSTRACT
Adenosine deaminases acting on RNA (ADARs) are the primary factors underlying adenosine to inosine (A-to-I) editing in metazoans. Here we report the first global study of ADAR1-RNA interaction in human cells using CLIP-seq. A large number of CLIP sites are observed in Alu repeats, consistent with ADAR1's function in RNA editing. Surprisingly, thousands of other CLIP sites are located in non-Alu regions, revealing functional and biophysical targets of ADAR1 in the regulation of alternative 3' UTR usage and miRNA biogenesis. We observe that binding of ADAR1 to 3' UTRs precludes binding by other factors, causing 3' UTR lengthening. Similarly, ADAR1 interacts with DROSHA and DGCR8 in the nucleus and possibly out-competes DGCR8 in primary miRNA binding, which enhances mature miRNA expression. These functions are dependent on ADAR1's editing activity, at least for a subset of targets. Our study unfolds a broad landscape of the functional roles of ADAR1.

No MeSH data available.