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Molecular and Cellular Biology, April 2009, p. 2155-2167, Vol. 29, No. 8
0270-7306/09/$08.00+0     doi:10.1128/MCB.01325-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of a Cytoplasmic Complex That Adds a Cap onto 5'-Monophosphate RNA {triangledown} ,§

Yuichi Otsuka,1 Nancy L. Kedersha,2 and Daniel R. Schoenberg1*

Department of Molecular and Cellular Biochemistry, Center for RNA Biology and the Comprehensive Cancer Center, the Ohio State University, Columbus, Ohio 43210,1 Division of Rheumatology and Immunology, Brigham and Women's Hospital, Boston, Massachusetts 021152

Received 19 August 2008/ Returned for modification 25 September 2008/ Accepted 2 February 2009

Endonuclease decay of nonsense-containing β-globin mRNA in erythroid cells generates 5'-truncated products that were reported previously to have a cap or caplike structure. We confirmed that this 5' modification is indistinguishable from the cap on full-length mRNA, and Western blotting, immunoprecipitation, and active-site labeling identified a population of capping enzymes in the cytoplasm of erythroid and nonerythroid cells. Cytoplasmic capping enzyme sediments in a 140-kDa complex that contains a kinase which, together with capping enzyme, converts 5'-monophosphate RNA into 5'-GpppX RNA. Capping enzyme shows diffuse and punctate staining throughout the cytoplasm, and its staining does not overlap with P bodies or stress granules. Expression of inactive capping enzyme in a form that is restricted to the cytoplasm reduced the ability of cells to recover from oxidative stress, thus supporting a role for capping in the cytoplasm and suggesting that some mRNAs may be stored in an uncapped state.

* Corresponding author. Mailing address: Department of Molecular and Cellular Biochemistry, the Ohio State University, 1645 Neil Ave., Columbus, OH 43210-1218. Phone: (614) 688-3012. Fax: (614) 292-4118. E-mail: schoenberg.3{at}osu.edu

{triangledown} Published ahead of print on 17 February 2009.

§ Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, April 2009, p. 2155-2167, Vol. 29, No. 8
0270-7306/09/$08.00+0     doi:10.1128/MCB.01325-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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