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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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Analysis of hMTr1 and hMTr2 mutants. In vitro transcribed RNA-GG molecules with 32P-labeled cap0 (A) or cap01 (B) structures were incubated with indicated enzymes [wild-type (hMTr1, hMTr2), alanine-substituted variants of hMTr1 (K239A, D365A, K404A) and hMTr2 (K117A, D235A, K275A) or truncated forms of hMTr2 (1–530, 1–430)] in the presence of SAM. Purified product RNA was digested with nuclease P1 (A) or RNase T2 (B). Digestion products were resolved on a 21% polyacrylamide/8 M urea gel and visualized by autoradiography. Asterisks indicate positions of 32P-labeled phosphates.
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Figure 3: Analysis of hMTr1 and hMTr2 mutants. In vitro transcribed RNA-GG molecules with 32P-labeled cap0 (A) or cap01 (B) structures were incubated with indicated enzymes [wild-type (hMTr1, hMTr2), alanine-substituted variants of hMTr1 (K239A, D365A, K404A) and hMTr2 (K117A, D235A, K275A) or truncated forms of hMTr2 (1–530, 1–430)] in the presence of SAM. Purified product RNA was digested with nuclease P1 (A) or RNase T2 (B). Digestion products were resolved on a 21% polyacrylamide/8 M urea gel and visualized by autoradiography. Asterisks indicate positions of 32P-labeled phosphates.

Mentions: To verify the predicted K-D-K-active sites of the hMTr1 and hMTr2 enzymes, site-directed mutagenesis was performed to generate protein variants with individual residues substituted by alanine. In vitro transcribed RNA-GG with 32P-labeled cap0 or cap01 structures were incubated with the purified protein variants in the presence of SAM. The reaction products were digested with nuclease P1 or RNase T2 and separated on a polyacrylamide gel. None of the variants exhibited MTase activity (Figure 3), indicating that each residue of the K-D-K triad is essential for the activity of both hMTr1 (Figure 3A, lanes 4–6) and hMTr2 (Figure 3B, lanes 3–5).Figure 3.


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)

Analysis of hMTr1 and hMTr2 mutants. In vitro transcribed RNA-GG molecules with 32P-labeled cap0 (A) or cap01 (B) structures were incubated with indicated enzymes [wild-type (hMTr1, hMTr2), alanine-substituted variants of hMTr1 (K239A, D365A, K404A) and hMTr2 (K117A, D235A, K275A) or truncated forms of hMTr2 (1–530, 1–430)] in the presence of SAM. Purified product RNA was digested with nuclease P1 (A) or RNase T2 (B). Digestion products were resolved on a 21% polyacrylamide/8 M urea gel and visualized by autoradiography. Asterisks indicate positions of 32P-labeled phosphates.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113572&req=5

Figure 3: Analysis of hMTr1 and hMTr2 mutants. In vitro transcribed RNA-GG molecules with 32P-labeled cap0 (A) or cap01 (B) structures were incubated with indicated enzymes [wild-type (hMTr1, hMTr2), alanine-substituted variants of hMTr1 (K239A, D365A, K404A) and hMTr2 (K117A, D235A, K275A) or truncated forms of hMTr2 (1–530, 1–430)] in the presence of SAM. Purified product RNA was digested with nuclease P1 (A) or RNase T2 (B). Digestion products were resolved on a 21% polyacrylamide/8 M urea gel and visualized by autoradiography. Asterisks indicate positions of 32P-labeled phosphates.
Mentions: To verify the predicted K-D-K-active sites of the hMTr1 and hMTr2 enzymes, site-directed mutagenesis was performed to generate protein variants with individual residues substituted by alanine. In vitro transcribed RNA-GG with 32P-labeled cap0 or cap01 structures were incubated with the purified protein variants in the presence of SAM. The reaction products were digested with nuclease P1 or RNase T2 and separated on a polyacrylamide gel. None of the variants exhibited MTase activity (Figure 3), indicating that each residue of the K-D-K triad is essential for the activity of both hMTr1 (Figure 3A, lanes 4–6) and hMTr2 (Figure 3B, lanes 3–5).Figure 3.

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