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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.


Related in: MedlinePlus

CLIP-Seq identifies ADAR1 binding sites in >10,000 human genes(a) Reproducibility of ADAR1 CLIP tags using two different antibodies. Each dot in the scatter plot represents log2 enrichment relative to the background abundance measured by polyA+ RNA-Seq for a RefSeq transcript. (b) CLIP tag distribution in the 3' UTR of the PSMB gene. The secondary structure of this region is shown as predicted by RNAfold. The number of CLIP tags is shown for each corresponding position in the folded structure, together with the location of two Alu sequences (inverted-repeats). Known editing sites (DARNED database) are labeled with red dots. (c) Genomic distribution of reproducible ADAR1 CLIP sites. Similar distribution of nucleotides in the entire transcriptome is shown as a reference. (d) Alignment of CLIP reads to the consensus Alu sequence. The CLIP tag density was normalized against expected tag density obtained from simulated reads to represent overall sequence enrichment of all the relevant Alus. Alignment to the sense Alu consensus and antisense Alu consensus was carried out separately. Given their strand-specific nature, CLIP reads were aligned to either the sense or antisense Alu unambiguously. The motif most enriched in ADAR1 CLIP tags is shown (based on an independent motif search within CLIP clusters by MEME), which is located in the sense Alu as labeled by the red bar. The motif enriched near editing sites in U87MG cells discovered previously 21 is also shown for comparison purpose.
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Figure 1: CLIP-Seq identifies ADAR1 binding sites in >10,000 human genes(a) Reproducibility of ADAR1 CLIP tags using two different antibodies. Each dot in the scatter plot represents log2 enrichment relative to the background abundance measured by polyA+ RNA-Seq for a RefSeq transcript. (b) CLIP tag distribution in the 3' UTR of the PSMB gene. The secondary structure of this region is shown as predicted by RNAfold. The number of CLIP tags is shown for each corresponding position in the folded structure, together with the location of two Alu sequences (inverted-repeats). Known editing sites (DARNED database) are labeled with red dots. (c) Genomic distribution of reproducible ADAR1 CLIP sites. Similar distribution of nucleotides in the entire transcriptome is shown as a reference. (d) Alignment of CLIP reads to the consensus Alu sequence. The CLIP tag density was normalized against expected tag density obtained from simulated reads to represent overall sequence enrichment of all the relevant Alus. Alignment to the sense Alu consensus and antisense Alu consensus was carried out separately. Given their strand-specific nature, CLIP reads were aligned to either the sense or antisense Alu unambiguously. The motif most enriched in ADAR1 CLIP tags is shown (based on an independent motif search within CLIP clusters by MEME), which is located in the sense Alu as labeled by the red bar. The motif enriched near editing sites in U87MG cells discovered previously 21 is also shown for comparison purpose.

Mentions: To elucidate the function of ADAR1 on the genome-wide scale, we first obtained global binding patterns of this protein using CLIP-Seq 22 in human U87MG cells. In this cell type, ADAR1 is expressed at a medium to high level, while ADAR2 and ADAR3 are barely expressed 21. We constructed two libraries using two ADAR1 antibodies (Santa Cruz Biotechnologies). Both antibodies can recognize two isoforms of ADAR1 (p150 and p110) (Supplementary Fig. 1). From each CLIP library, more than 10 million reads were obtained with confident mapping to the human genome (Supplementary Table 1). To assess the reproducibility of the experiments, we examined the correlation of CLIP-Seq tag abundance between the two libraries precipitated with different antibodies. As shown in Fig. 1a, the two libraries yielded highly correlated results, suggesting that most of the CLIP tags reflect the common pool of ADAR1-interacting RNAs.


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)

CLIP-Seq identifies ADAR1 binding sites in >10,000 human genes(a) Reproducibility of ADAR1 CLIP tags using two different antibodies. Each dot in the scatter plot represents log2 enrichment relative to the background abundance measured by polyA+ RNA-Seq for a RefSeq transcript. (b) CLIP tag distribution in the 3' UTR of the PSMB gene. The secondary structure of this region is shown as predicted by RNAfold. The number of CLIP tags is shown for each corresponding position in the folded structure, together with the location of two Alu sequences (inverted-repeats). Known editing sites (DARNED database) are labeled with red dots. (c) Genomic distribution of reproducible ADAR1 CLIP sites. Similar distribution of nucleotides in the entire transcriptome is shown as a reference. (d) Alignment of CLIP reads to the consensus Alu sequence. The CLIP tag density was normalized against expected tag density obtained from simulated reads to represent overall sequence enrichment of all the relevant Alus. Alignment to the sense Alu consensus and antisense Alu consensus was carried out separately. Given their strand-specific nature, CLIP reads were aligned to either the sense or antisense Alu unambiguously. The motif most enriched in ADAR1 CLIP tags is shown (based on an independent motif search within CLIP clusters by MEME), which is located in the sense Alu as labeled by the red bar. The motif enriched near editing sites in U87MG cells discovered previously 21 is also shown for comparison purpose.
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Related In: Results  -  Collection

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Figure 1: CLIP-Seq identifies ADAR1 binding sites in >10,000 human genes(a) Reproducibility of ADAR1 CLIP tags using two different antibodies. Each dot in the scatter plot represents log2 enrichment relative to the background abundance measured by polyA+ RNA-Seq for a RefSeq transcript. (b) CLIP tag distribution in the 3' UTR of the PSMB gene. The secondary structure of this region is shown as predicted by RNAfold. The number of CLIP tags is shown for each corresponding position in the folded structure, together with the location of two Alu sequences (inverted-repeats). Known editing sites (DARNED database) are labeled with red dots. (c) Genomic distribution of reproducible ADAR1 CLIP sites. Similar distribution of nucleotides in the entire transcriptome is shown as a reference. (d) Alignment of CLIP reads to the consensus Alu sequence. The CLIP tag density was normalized against expected tag density obtained from simulated reads to represent overall sequence enrichment of all the relevant Alus. Alignment to the sense Alu consensus and antisense Alu consensus was carried out separately. Given their strand-specific nature, CLIP reads were aligned to either the sense or antisense Alu unambiguously. The motif most enriched in ADAR1 CLIP tags is shown (based on an independent motif search within CLIP clusters by MEME), which is located in the sense Alu as labeled by the red bar. The motif enriched near editing sites in U87MG cells discovered previously 21 is also shown for comparison purpose.
Mentions: To elucidate the function of ADAR1 on the genome-wide scale, we first obtained global binding patterns of this protein using CLIP-Seq 22 in human U87MG cells. In this cell type, ADAR1 is expressed at a medium to high level, while ADAR2 and ADAR3 are barely expressed 21. We constructed two libraries using two ADAR1 antibodies (Santa Cruz Biotechnologies). Both antibodies can recognize two isoforms of ADAR1 (p150 and p110) (Supplementary Fig. 1). From each CLIP library, more than 10 million reads were obtained with confident mapping to the human genome (Supplementary Table 1). To assess the reproducibility of the experiments, we examined the correlation of CLIP-Seq tag abundance between the two libraries precipitated with different antibodies. As shown in Fig. 1a, the two libraries yielded highly correlated results, suggesting that most of the CLIP tags reflect the common pool of ADAR1-interacting RNAs.

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.


Related in: MedlinePlus