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Distinct in vivo roles for double-stranded RNA-binding domains of the Xenopus RNA-editing enzyme ADAR1 in chromosomal targeting.

Doyle M, Jantsch MF - J. Cell Biol. (2003)

Bottom Line: Previously, we could show that Xenopus ADAR1 is associated with nascent transcripts on transcriptionally active lampbrush chromosomes, indicating that initial substrate binding and possibly editing itself occurs cotranscriptionally.Here, we demonstrate that chromosomal association depends solely on the three double-stranded RNA-binding domains (dsRBDs) found in the central part of ADAR1, but not on the Z-DNA-binding domain in the NH2 terminus nor the catalytic deaminase domain in the COOH terminus of the protein.Thus, our results not only prove the requirement of dsRBDs for chromosomal targeting, but also show that individual dsRBDs have distinct in vivo localization capabilities that may be important for initial substrate recognition and subsequent editing specificity.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Cell Biology and Genetics, Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria.

ABSTRACT
The RNA-editing enzyme adenosine deaminase that acts on RNA (ADAR1) deaminates adenosines to inosines in double-stranded RNA substrates. Currently, it is not clear how the enzyme targets and discriminates different substrates in vivo. However, it has been shown that the deaminase domain plays an important role in distinguishing various adenosines within a given substrate RNA in vitro. Previously, we could show that Xenopus ADAR1 is associated with nascent transcripts on transcriptionally active lampbrush chromosomes, indicating that initial substrate binding and possibly editing itself occurs cotranscriptionally. Here, we demonstrate that chromosomal association depends solely on the three double-stranded RNA-binding domains (dsRBDs) found in the central part of ADAR1, but not on the Z-DNA-binding domain in the NH2 terminus nor the catalytic deaminase domain in the COOH terminus of the protein. Most importantly, we show that individual dsRBDs are capable of recognizing different chromosomal sites in an apparently specific manner. Thus, our results not only prove the requirement of dsRBDs for chromosomal targeting, but also show that individual dsRBDs have distinct in vivo localization capabilities that may be important for initial substrate recognition and subsequent editing specificity.

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Northwestern analysis of dsRBDs constructs. The RNA-binding assay of bacterially expressed dsRBD-GST fusion proteins was performed with rI/rC (A). In parallel, the same extracts were run on a gel and stained with Coomassie brilliant blue to allow quantification of the recombinant protein (B). The ratio of signal to protein was quantified by laser densitometry and is depicted graphically (C). The number and type of dsRBDs are indicated. Also shown is the empty pGEX vector used as a negative control, whereas the second dsRBD of XlrbpA is included as a positive control. Of the single domains, dsRBD2 and dsRBD3 were found to be the best RNA binders. Constructs containing duplications of individual dsRBDs showed a higher affinity for rI/rC, whereas triplicating individual dsRBDs further enhanced this affinity.
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fig4: Northwestern analysis of dsRBDs constructs. The RNA-binding assay of bacterially expressed dsRBD-GST fusion proteins was performed with rI/rC (A). In parallel, the same extracts were run on a gel and stained with Coomassie brilliant blue to allow quantification of the recombinant protein (B). The ratio of signal to protein was quantified by laser densitometry and is depicted graphically (C). The number and type of dsRBDs are indicated. Also shown is the empty pGEX vector used as a negative control, whereas the second dsRBD of XlrbpA is included as a positive control. Of the single domains, dsRBD2 and dsRBD3 were found to be the best RNA binders. Constructs containing duplications of individual dsRBDs showed a higher affinity for rI/rC, whereas triplicating individual dsRBDs further enhanced this affinity.

Mentions: To evaluate the RNA-binding ability of these constructs in vitro and to compare them with each other, all dsRBD combinations (single, duplications, and triplications) were expressed as GST fusion proteins and tested in Northwestern assays using rI/rC as a probe. At the same time, the second dsRBD of Xenopus laevis RNA-binding protein A (XlrbpA) was used as a positive control for RNA-binding as this domain binds RNA with very high affinity (Krovat and Jantsch, 1996) and had been used previously as an internal standard to quantify the RNA-binding capacity of the individual dsRBDs of Xenopus ADAR1 (Brooks et al., 1998). To determine the amount of recombinant protein in the extracts, a second gel was run in parallel and stained with Coomassie brilliant blue. The autoradiogram and Coomassie-stained gels were quantified using laser densitometry, and the amount of RNA bound was normalized to the amount of recombinant protein present in the extracts (Fig. 4) .


Distinct in vivo roles for double-stranded RNA-binding domains of the Xenopus RNA-editing enzyme ADAR1 in chromosomal targeting.

Doyle M, Jantsch MF - J. Cell Biol. (2003)

Northwestern analysis of dsRBDs constructs. The RNA-binding assay of bacterially expressed dsRBD-GST fusion proteins was performed with rI/rC (A). In parallel, the same extracts were run on a gel and stained with Coomassie brilliant blue to allow quantification of the recombinant protein (B). The ratio of signal to protein was quantified by laser densitometry and is depicted graphically (C). The number and type of dsRBDs are indicated. Also shown is the empty pGEX vector used as a negative control, whereas the second dsRBD of XlrbpA is included as a positive control. Of the single domains, dsRBD2 and dsRBD3 were found to be the best RNA binders. Constructs containing duplications of individual dsRBDs showed a higher affinity for rI/rC, whereas triplicating individual dsRBDs further enhanced this affinity.
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Related In: Results  -  Collection

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fig4: Northwestern analysis of dsRBDs constructs. The RNA-binding assay of bacterially expressed dsRBD-GST fusion proteins was performed with rI/rC (A). In parallel, the same extracts were run on a gel and stained with Coomassie brilliant blue to allow quantification of the recombinant protein (B). The ratio of signal to protein was quantified by laser densitometry and is depicted graphically (C). The number and type of dsRBDs are indicated. Also shown is the empty pGEX vector used as a negative control, whereas the second dsRBD of XlrbpA is included as a positive control. Of the single domains, dsRBD2 and dsRBD3 were found to be the best RNA binders. Constructs containing duplications of individual dsRBDs showed a higher affinity for rI/rC, whereas triplicating individual dsRBDs further enhanced this affinity.
Mentions: To evaluate the RNA-binding ability of these constructs in vitro and to compare them with each other, all dsRBD combinations (single, duplications, and triplications) were expressed as GST fusion proteins and tested in Northwestern assays using rI/rC as a probe. At the same time, the second dsRBD of Xenopus laevis RNA-binding protein A (XlrbpA) was used as a positive control for RNA-binding as this domain binds RNA with very high affinity (Krovat and Jantsch, 1996) and had been used previously as an internal standard to quantify the RNA-binding capacity of the individual dsRBDs of Xenopus ADAR1 (Brooks et al., 1998). To determine the amount of recombinant protein in the extracts, a second gel was run in parallel and stained with Coomassie brilliant blue. The autoradiogram and Coomassie-stained gels were quantified using laser densitometry, and the amount of RNA bound was normalized to the amount of recombinant protein present in the extracts (Fig. 4) .

Bottom Line: Previously, we could show that Xenopus ADAR1 is associated with nascent transcripts on transcriptionally active lampbrush chromosomes, indicating that initial substrate binding and possibly editing itself occurs cotranscriptionally.Here, we demonstrate that chromosomal association depends solely on the three double-stranded RNA-binding domains (dsRBDs) found in the central part of ADAR1, but not on the Z-DNA-binding domain in the NH2 terminus nor the catalytic deaminase domain in the COOH terminus of the protein.Thus, our results not only prove the requirement of dsRBDs for chromosomal targeting, but also show that individual dsRBDs have distinct in vivo localization capabilities that may be important for initial substrate recognition and subsequent editing specificity.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Cell Biology and Genetics, Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria.

ABSTRACT
The RNA-editing enzyme adenosine deaminase that acts on RNA (ADAR1) deaminates adenosines to inosines in double-stranded RNA substrates. Currently, it is not clear how the enzyme targets and discriminates different substrates in vivo. However, it has been shown that the deaminase domain plays an important role in distinguishing various adenosines within a given substrate RNA in vitro. Previously, we could show that Xenopus ADAR1 is associated with nascent transcripts on transcriptionally active lampbrush chromosomes, indicating that initial substrate binding and possibly editing itself occurs cotranscriptionally. Here, we demonstrate that chromosomal association depends solely on the three double-stranded RNA-binding domains (dsRBDs) found in the central part of ADAR1, but not on the Z-DNA-binding domain in the NH2 terminus nor the catalytic deaminase domain in the COOH terminus of the protein. Most importantly, we show that individual dsRBDs are capable of recognizing different chromosomal sites in an apparently specific manner. Thus, our results not only prove the requirement of dsRBDs for chromosomal targeting, but also show that individual dsRBDs have distinct in vivo localization capabilities that may be important for initial substrate recognition and subsequent editing specificity.

Show MeSH