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Molecular and Cellular Biology, April 2005, p. 2644-2649, Vol. 25, No. 7
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.7.2644-2649.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Human mRNA Cap Methyltransferase: Alternative Nuclear Localization Signal Motifs Ensure Nuclear Localization Required for Viability{dagger}

Beth Shafer,{ddagger} Chun Chu,{ddagger} and Aaron J. Shatkin*

Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey

Received 30 September 2004/ Returned for modification 18 November 2004/ Accepted 21 December 2004

A characteristic feature of gene expression in eukaryotes is the addition of a 5'-terminal 7-methylguanine cap (m7GpppN) to nascent pre-mRNAs in the nucleus catalyzed by capping enzyme and cap methyltransferase. Small interfering RNA (siRNA) knockdown of cap methyltransferase in HeLa cells resulted in apoptosis as measured by terminal deoxynucleotidyltransferase-mediated dUTP-tetramethylrhodamine nick end labeling assay, demonstrating the importance of mRNA 5'-end methylation for mammalian cell viability. Nuclear localization of cap methyltransferase is mediated by interaction with importin-{alpha}, which facilitates its transport and selective binding to transcripts containing 5'-terminal GpppN. The methyltransferase 96-144 region has been shown to be necessary for importin binding, and N-terminal fusion of this sequence to nonnuclear proteins proved sufficient for nuclear localization. The targeting sequence was narrowed to amino acids 120 to 129, including a required 126KRK. Although full-length methyltransferase (positions 1 to 476) contains the predicted nuclear localization signals 57RKRK, 80KKRK, 103KKRKR, and 194KKKR, mutagenesis studies confirmed functional motifs only at positions 80, 103, and the previously unrecognized 126KRK. All three motifs can act as alternative nu clear targeting signals. Expression of N-truncated cap methyltransferase (120 to 476) restored viability of methyltransferase siRNA knocked-down cells. However, an enzymatically active 144-476 truncation mutant missing the three nuclear localization signals was mostly cytoplasmic and ineffective in preventing siRNA-induced loss of viability.

* Corresponding author. Mailing address: Center for Advanced Biotechnology and Medicine, 679 Hoes Ln., Piscataway, NJ 08854. Phone: (732) 235-5311. Fax: (732) 235-5318. E-mail: shatkin{at}cabm.rutgers.edu.

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

{ddagger} B.S. and C.C. contributed equally.

Molecular and Cellular Biology, April 2005, p. 2644-2649, Vol. 25, No. 7
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.7.2644-2649.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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