ARD-1 cDNA from Human Cells Encodes a Site-specific Single-strand Endoribonuclease That Functionally Resembles Escherichia coli RNase E*
- From the Department of Genetics, Stanford University School of Medicine, Stanford, California 94305-5120
Abstract
The human ARD-1 (activator of RNA decay) cDNA sequence can rescue mutations in the Escherichia coli rne gene, which specifies the essential endoribonuclease RNase E, resulting in RNase E-like cleavages in vivo inrne-defective bacteria and in vitro in extracts isolated from these cells (Wang, M., and Cohen, S. N. (1994)Proc. Natl. Acad. Sci. U. S. A. 91, 10591–10595). Recent studies indicate that the 13.3-kDa protein encoded by ARD-1cDNA is almost identical to the carboxyl-terminal end of the bovine protein NIPP-1, a nuclear inhibitor of protein phosphatase 1; separate transcripts formed by alternative splicing are proposed to encode the discrete ARD-1 and combined ARD-1/NIPP-1 products (Van Eynde, A., Wera, S., Beullens, M., Torrekens, S., Van Leuven, F., Stalmans, W., and Bollens, M. (1995) J. Biol. Chem. 270, 28068–28074). Here we show that affinity column-purified protein encoded by humanARD-1 cDNA in E. coli is a site-specific Mg2+-dependent endoribonuclease that bindsin vitro to RNase E substrates, cleaves RNA at the same sites as RNase E, and, like RNase E, generates 5′ phosphate termini at sites of cleavage. Our results indicate that the ARD-1 peptide can function as a ribonucleolytic analog of E. coli RNase E as well as a domain of the protein phosphatase inhibitor, NIPP-1.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grant GM27241 (to S. N. C.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ These authors contributed equally to this work.
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↵§ Present address: Unidad de Investigación, Hospital de la Candelaria, 38010 Santa Cruz de Tenerife, Spain.
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↵¶ Supported in part by Predoctoral Training Grant EYO7106 from NEI, National Institutes of Health. Present address: Hyseq, Inc., 670 Almanor Ave., Sunnyvale, CA 94086.
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↵‖ To whom correspondence and reprint requests should be addressed: Dept. of Genetics, Rm. M-320, Stanford Medical Center, Stanford, CA 94305. Tel.: 415-723-5315; Fax: 415-725-1536; E-mail:sncohen{at}forsythe.stanford.edu.
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↵1 P. Cohen, personal communication.
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↵2 The abbreviations used are: bp, base pair(s); PCR, polymerase chain reaction; IPTG, isopropyl-1-thio-β-d-galactopyranoside; nt, nucleotide(s).
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- Received December 24, 1996.
- Revision received March 25, 1997.
