Detection of dsRNA-binding domains in RNA helicase A and<i>Drosophila</i>maleless: implications for monomeric RNA helicases

Gibson, Toby J.; Thompson, Julie

doi:10.1093/nar/22.13.2552

Cited by 92 publications

(78 citation statements)

References 37 publications

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“…This region would contain both dsRBDs that have been attributed to the amino acid residues 3-72 and 180 -253, respectively (Fig. 7) (6). It was surprising that full-length NDH II reacted more weakly with the antihistidine antibody than the COOH-terminally deleted form (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…At the amino terminus there are two additional binding domains (dsRBD I and II) that might provide specificity for RNA (6). Similar motifs are conserved among a group of dsRNA-binding proteins, such as dsRNA-dependent protein kinase (DAI) (7), dsRNA-specific adenosine deaminase (DRADA) (8), Escherichia coli RNase III (9), the Drosophila staufen protein, the Xenopus laevis RNA-binding protein Xlrbpa, the human transactivator region binding protein (10,11), and the vaccinia virus E3L protein (12).…”

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confidence: 99%

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Domain Structure of Human Nuclear DNA Helicase II (RNA Helicase A)

Zhang¹,

Grosse²

1997

Journal of Biological Chemistry

110

125

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Full-length human nuclear DNA helicase II (NDH II) was cloned and overexpressed in a baculovirus-derived expression system. Recombinant NDH II unwound both DNA and RNA. Limited tryptic digestion produced active helicases with molecular masses of 130 and 100 kDa. The 130-kDa helicase missed a glycine-rich domain (RGG-box) at the carboxyl terminus, while the 100-kDa form missed both its double-stranded RNA binding domains (dsRBDs) at the amino terminus and its RGG-box. Hence, the dsRBDs and the RGG-box were dispensable for unwinding. On the other hand, the isolated DEXH core alone could neither hydrolyze ATP nor unwind nucleic acids. These enzymatic activities were not regained by fusing a complete COOH or NH 2 terminus to the helicase core. Hence, an active helicase required part of the NH 2 terminus, the DEXH core, and a C-terminal extension of the core. Both dsRBDs and the RGGbox were bacterially expressed as glutathione S-transferase fusion proteins. The two dsRBDs had a strong affinity to double-stranded RNA and cooperated upon RNA binding, while the RGG-box bound preferentially to single-stranded DNA. A model is suggested in which the flanking domains influence and regulate the unwinding properties of NDH II.Nuclear DNA helicase II (NDH II) 1 was originally isolated from calf thymus by assaying its DNA unwinding activity (1). Subsequently, it was shown that NDH II also contains RNA helicase activity (2). The cDNA sequence of NDH II (3) revealed a high homology to two previously known proteins, namely human RNA helicase A (4) and the Drosophila "maleless" protein (MLE) (5). From the cDNA sequences it was also deduced that the three proteins belong to the superfamily of DEXH helicases, all members of which possess seven conserved helicase motifs in the putative catalytically active core. The presence of the DEXH helicase core domain suggests a function in RNA and/or DNA unwinding for all three members of this superfamily. The core contains an ATP binding site with the consensus sequence GCGKT (A site) and FILDD (B site) in motif

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Domain Structure of Human Nuclear DNA Helicase II (RNA Helicase A)

Zhang¹,

Grosse²

1997

Journal of Biological Chemistry

110

125

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“…Comparison of the RNase III amino acid sequence with the protein databases showed a double-stranded RNA-binding domain (dsRBD) common with a large group of double-stranded RNA-binding proteins (Court, 1993). The dsRBD has since been found to be a universally conserved oligopeptide module attached to a variety of regulatory and structural proteins (Green and Mathews, 1992;St Johnston et al, 1992;Gibson et al, 1993;Gibson and Thompson, 1994;O'Connell et al, 1995;Kharrat et al, 1995;Nicholson, 1996). The putative RNA-binding domain at the carboxy-terminus of RNase III has since been shown to maintain a canonical dsRBD conformation even when separated from the whole enzyme (Kharrat et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

Genetic uncoupling of the dsRNA‐binding and RNA cleavage activities of the Escherichia coli endoribonuclease RNase III — the effect of dsRNA binding on gene expression

Dasgupta

Fernández

Kameyama

et al. 1998

Molecular Microbiology

100

108

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SummaryRNase III, a double-stranded RNA-specific endonuclease, is proposed to be one of Escherichia coli 's global regulators because of its ability to affect the expression of a large number of unrelated genes by influencing post-transcriptional control of mRNA stability or mRNA translational efficiency. Here, we describe the phenotypes of bacteria carrying point mutations in rnc, the gene encoding RNase III. The substrate recognition and RNA-processing properties of mutant proteins were analysed in vivo by measuring expression from known RNase III-modulated genes and in vitro from the proteins' binding and cleavage activities on known double-stranded RNA substrates. Our results show that although the point mutation rnc70 exhibited all the usual rnc null-like phenotypes, unlike other mutations, it was dominant over the wild-type allele. Multicopy expression of rnc70 could suppress a lethal phenotype of the wild-type rnc allele in a certain genetic background; it could also inhibit the RNase III-mediated activation of N gene translation by competing for the RNA-binding site of the wild-type endonuclease. The mutant protein failed to cleave the standard RNase III substrates in vitro but exhibited an affinity for double-stranded RNA when passed through poly(rI):poly(rC) columns. Filter binding and gel-shift assays with purified Rnc70 showed that the mutant protein binds to known RNase III mRNA substrates in a site-specific manner. In vitro processing reactions with purified enzyme and labelled RNA showed that the in vivo dominant effect of the mutant enzyme over the wild-type was not necessarily caused by formation of mixed dimers. Thus, the rnc70 mutation generates a mutant RNase III with impaired endonucleolytic activity but without blocking its ability to recognize and bind double-stranded RNA substrates.

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“…The amino terminal portion of the MLE protein contains two copies of a double stranded RNA binding motif (St. Johnston et al 1992;Gibson & Thompson 1994). The central portion of the MLE protein contains a domain similar to the DEAD box family of ATPase and helicase proteins .…”

Section: Introductionmentioning

confidence: 99%

RNA‐dependent association of the Drosophila maleless protein with the male X chromosome

1996

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Background: Dosage compensation results in equivalent X-linked gene expression in males (XY) and females (XX). In Drosophila, both X chromosomes are active in females, and the single male X must double its transcriptional activity to allow male development. Four proteins (encoded by the malespecific lethal genes) are required for dosage compensation and associate with the X chromosome in males but not in females.

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Detection of dsRNA-binding domains in RNA helicase A andDrosophilamaleless: implications for monomeric RNA helicases

Abstract: ABSTRACT

Cited by 92 publications

References 37 publications

Domain Structure of Human Nuclear DNA Helicase II (RNA Helicase A)

Domain Structure of Human Nuclear DNA Helicase II (RNA Helicase A)

Genetic uncoupling of the dsRNA‐binding and RNA cleavage activities of the Escherichia coli endoribonuclease RNase III — the effect of dsRNA binding on gene expression

RNA‐dependent association of the Drosophila maleless protein with the male X chromosome

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