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. 2005 Apr;25(7):2644-9.
doi: 10.1128/MCB.25.7.2644-2649.2005.

Human mRNA cap methyltransferase: alternative nuclear localization signal motifs ensure nuclear localization required for viability

Beth Shafer  1 , Chun Chu, Aaron J Shatkin
Affiliations

Human mRNA cap methyltransferase: alternative nuclear localization signal motifs ensure nuclear localization required for viability

Beth Shafer et al. Mol Cell Biol. 2005 Apr.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Intracellular localization of chimeric GFP-tagged proteins. HeLa cells were transfected with plasmids for expression of RTP-GFP (A), MT-GFP (B), 96-144MT-RTP-GFP (C), 96-144MT-GFP (D), RAF1-GFP (E), or 96-144MT-RAF1-GFP (F) and 36 h later were examined by fluorescence microscopy (Fl) and Hoechst nuclear staining (Nu) as described in Materials and Methods.
FIG. 2.
FIG. 2.
Loss of nuclear targeting by mutation of 126 KRK in MT-RTP-GFP. Nuclear targeting of 120-129MT-RTP-GFP (A) and 96-129MT-RTP-GFP with 103KKRKR mutagenized to all alanines (B). Cytoplasmic localization of 120-129MT-RTP-GFP containing K126A (C), R127I (D), or K128A (E). Fl, fluorescence microscopy; Nu, Hoechst nuclear staining.
FIG. 3.
FIG. 3.
Binding of MT-RTP-GFP chimeras to GST-Impα. (A) MT input, GST bound, and GST-Impα bound (lanes 1, 2, and 3); input (25% in all cases) and bound are shown in the even and odd lanes, respectively, for GFP (lanes 4 and 5), RTP-GFP (lanes 6 and 7), 96-144MT-RTP-GFP (lanes 8 and 9), 96-129MT-RTP-GFP (lanes 10 and 11), 120-144MT-RTP-GFP (lanes 12 and 13), and 120-129MT-RTP-GFP (lanes 14 and 15). (B) The fraction of each MT-RTP-GFP chimera that bound to GST-Impα was determined by phosphorimager analysis and then normalized relative to the binding of full-length MT-GFP. There was a direct, linear relationship between the relative binding and the number of MT residues fused to RTP-GFP (r2 = 0.9835).
FIG. 4.
FIG. 4.
Effects of N-terminal truncation and mutation on MT localization. HeLa cells were examined 36 h after transfection with 96-476MT-GFP (A), 120-476MT-GFP (B), 130-476MT-GFP (C), 120-476(R127A)MT-GFP (D), 96-476(R127I)MT-GFP (E), 96-476(103-107A)MT-GFP (F), or 96-476(103-107A/R127I)MT-GFP (G). Fl, fluorescence microscopy; Nu, Hoechst nuclear staining.
FIG. 5.
FIG. 5.
Nuclear targeting of MT NLS mutants. MT triple mutants MT(57-60A, 103-107A, R127I)-GFP (A) and MT(80-83A, 103-107A, R127I)-GFP (B) were expressed in transfected HeLa cells. (C) Summary showing that MT sequence 126KRK is an NLS in addition to two other putative NLS motifs annotated by PSORTII Prediction, 80KKRK and 103KKRKR, while the predicted 57RKRK and 194KKKR motifs are not NLSs. Fl, fluorescence microscopy; Nu, Hoechst nuclear staining.
FIG. 6.
FIG. 6.
MT siRNA effect on MT-GFP expression. HeLa cells were transfected with MT-GFP alone (A) or also with MT317 mismatch siRNA (B) and MT317 siRNA (C) and examined by fluorescence microscopy.
FIG. 7.
FIG. 7.
Prevention of apoptosis by nuclearly localized MT-GFP constructs. TUNEL staining of untreated HeLa cells (A), mock-transfected cells (B), or cells transfected with MT317 mismatch (C), MT1085 (D), or MT317 (E), followed by transfection of full-length MT (F), the nuclear-targeted MT 120-476 truncation mutant (G), or the cytoplasmically localized MT 144-476 mutant (H).
FIG. 8.
FIG. 8.
Association of nuclearly localized MT with Pol II. HeLa cells were transfected with the indicated constructs, cross-linked with dithiobis[succinimidylpropionate], and immunoprecipitated with anti-Pol II. Cell lysates (L) and precipitates (P) were analyzed by Western blotting with anti-GFP.
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