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2'-O-ribose methylation of cap2 in human: function and evolution in a horizontally mobile family.

Werner M, Purta E, Kaminska KH, Cymerman IA, Campbell DA, Mittra B, Zamudio JR, Sturm NR, Jaworski J, Bujnicki JM - Nucleic Acids Res. (2011)

Bottom Line: The hMTr2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear hMTr1.The 2'-O-ribose RNA cap methyltransferases are present in varying combinations in most eukaryotic and many viral genomes.With the capping enzymes in hand their biological purpose can be ascertained.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.

ABSTRACT
The 5' cap of human messenger RNA consists of an inverted 7-methylguanosine linked to the first transcribed nucleotide by a unique 5'-5' triphosphate bond followed by 2'-O-ribose methylation of the first and often the second transcribed nucleotides, likely serving to modify efficiency of transcript processing, translation and stability. We report the validation of a human enzyme that methylates the ribose of the second transcribed nucleotide encoded by FTSJD1, henceforth renamed HMTR2 to reflect function. Purified recombinant hMTr2 protein transfers a methyl group from S-adenosylmethionine to the 2'-O-ribose of the second nucleotide of messenger RNA and small nuclear RNA. Neither N(7) methylation of the guanosine cap nor 2'-O-ribose methylation of the first transcribed nucleotide are required for hMTr2, but the presence of cap1 methylation increases hMTr2 activity. The hMTr2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear hMTr1. The details of how and why specific transcripts undergo modification with these ribose methylations remains to be elucidated. The 2'-O-ribose RNA cap methyltransferases are present in varying combinations in most eukaryotic and many viral genomes. With the capping enzymes in hand their biological purpose can be ascertained.

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Both hMTr1 and hMTr2 have a nuclear presence. MCF7 cells were transfected with GW1-myc-hMTr1, GW1-myc-hMTr2 or EGFP-C1 (control plasmid). After 24 h, cells were fixed and stained with anti-myc or anti-GFP antibodies to visualize recombinant proteins (green). Nuclei were visualized with Hoechst 33258 staining (blue) and F-actin with phalloidin 568 (red). Images were acquired by confocal microscopy. (A) Single focal planes from the Z-stack at nucleus level are presented. (B) Cells transfected with GW1-myc-hMTr2 with respective orthogonal axis view to verify the labeling.
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Figure 5: Both hMTr1 and hMTr2 have a nuclear presence. MCF7 cells were transfected with GW1-myc-hMTr1, GW1-myc-hMTr2 or EGFP-C1 (control plasmid). After 24 h, cells were fixed and stained with anti-myc or anti-GFP antibodies to visualize recombinant proteins (green). Nuclei were visualized with Hoechst 33258 staining (blue) and F-actin with phalloidin 568 (red). Images were acquired by confocal microscopy. (A) Single focal planes from the Z-stack at nucleus level are presented. (B) Cells transfected with GW1-myc-hMTr2 with respective orthogonal axis view to verify the labeling.

Mentions: Cellular localization of hMTr2 was assayed in situ by immunostaining of epitope-tagged protein. MCF7 cells were transfected with GW1-myc-hMTr2 or control plasmid EGFP-C1 that distributed throughout the cell. In parallel, hMTr1 was examined using GW1-myc-hMTr1. Twenty-four hours post-transfection, cells were fixed and immunostained for the overexpressed protein. The myc-hMTr2 was present in both the cytoplasm and the nucleus (Figure 5). Verification of labeling was done by checking the orthogonal axis views (Figure 5B). The unit volume ratio of myc-hMTr2 signal in the nucleoplasm versus cytosol was 1.25, comparable with the 1.45 ratio detected in cells expressing EGFP that was spread by passive diffusion. The myc-hMTr1 was confined to the nucleus (32,42) with almost no signal detectable in the cytosol. Both myc-hMTr1 and myc-hMTr2 were excluded from DNA-free nuclear bodies that are likely to represent nucleoli. Thus RNAs probably acquire the cap1 modification by hMTr1 during or shortly after transcription in the nucleus, followed by hMTr2 capping in either nucleus or after export to the cytoplasm. The mRNA and snRNAs may both serve as substrates for these enzymes, but their presentation may differ along with their divergent paths after acquisition of cap2.Figure 5.


2'-O-ribose methylation of cap2 in human: function and evolution in a horizontally mobile family.

Werner M, Purta E, Kaminska KH, Cymerman IA, Campbell DA, Mittra B, Zamudio JR, Sturm NR, Jaworski J, Bujnicki JM - Nucleic Acids Res. (2011)

Both hMTr1 and hMTr2 have a nuclear presence. MCF7 cells were transfected with GW1-myc-hMTr1, GW1-myc-hMTr2 or EGFP-C1 (control plasmid). After 24 h, cells were fixed and stained with anti-myc or anti-GFP antibodies to visualize recombinant proteins (green). Nuclei were visualized with Hoechst 33258 staining (blue) and F-actin with phalloidin 568 (red). Images were acquired by confocal microscopy. (A) Single focal planes from the Z-stack at nucleus level are presented. (B) Cells transfected with GW1-myc-hMTr2 with respective orthogonal axis view to verify the labeling.
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Figure 5: Both hMTr1 and hMTr2 have a nuclear presence. MCF7 cells were transfected with GW1-myc-hMTr1, GW1-myc-hMTr2 or EGFP-C1 (control plasmid). After 24 h, cells were fixed and stained with anti-myc or anti-GFP antibodies to visualize recombinant proteins (green). Nuclei were visualized with Hoechst 33258 staining (blue) and F-actin with phalloidin 568 (red). Images were acquired by confocal microscopy. (A) Single focal planes from the Z-stack at nucleus level are presented. (B) Cells transfected with GW1-myc-hMTr2 with respective orthogonal axis view to verify the labeling.
Mentions: Cellular localization of hMTr2 was assayed in situ by immunostaining of epitope-tagged protein. MCF7 cells were transfected with GW1-myc-hMTr2 or control plasmid EGFP-C1 that distributed throughout the cell. In parallel, hMTr1 was examined using GW1-myc-hMTr1. Twenty-four hours post-transfection, cells were fixed and immunostained for the overexpressed protein. The myc-hMTr2 was present in both the cytoplasm and the nucleus (Figure 5). Verification of labeling was done by checking the orthogonal axis views (Figure 5B). The unit volume ratio of myc-hMTr2 signal in the nucleoplasm versus cytosol was 1.25, comparable with the 1.45 ratio detected in cells expressing EGFP that was spread by passive diffusion. The myc-hMTr1 was confined to the nucleus (32,42) with almost no signal detectable in the cytosol. Both myc-hMTr1 and myc-hMTr2 were excluded from DNA-free nuclear bodies that are likely to represent nucleoli. Thus RNAs probably acquire the cap1 modification by hMTr1 during or shortly after transcription in the nucleus, followed by hMTr2 capping in either nucleus or after export to the cytoplasm. The mRNA and snRNAs may both serve as substrates for these enzymes, but their presentation may differ along with their divergent paths after acquisition of cap2.Figure 5.

Bottom Line: The hMTr2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear hMTr1.The 2'-O-ribose RNA cap methyltransferases are present in varying combinations in most eukaryotic and many viral genomes.With the capping enzymes in hand their biological purpose can be ascertained.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.

ABSTRACT
The 5' cap of human messenger RNA consists of an inverted 7-methylguanosine linked to the first transcribed nucleotide by a unique 5'-5' triphosphate bond followed by 2'-O-ribose methylation of the first and often the second transcribed nucleotides, likely serving to modify efficiency of transcript processing, translation and stability. We report the validation of a human enzyme that methylates the ribose of the second transcribed nucleotide encoded by FTSJD1, henceforth renamed HMTR2 to reflect function. Purified recombinant hMTr2 protein transfers a methyl group from S-adenosylmethionine to the 2'-O-ribose of the second nucleotide of messenger RNA and small nuclear RNA. Neither N(7) methylation of the guanosine cap nor 2'-O-ribose methylation of the first transcribed nucleotide are required for hMTr2, but the presence of cap1 methylation increases hMTr2 activity. The hMTr2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear hMTr1. The details of how and why specific transcripts undergo modification with these ribose methylations remains to be elucidated. The 2'-O-ribose RNA cap methyltransferases are present in varying combinations in most eukaryotic and many viral genomes. With the capping enzymes in hand their biological purpose can be ascertained.

Show MeSH