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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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hMTr2 activity and substrate requirements. Methyltransferase activity: In vitro transcribed RNA-GG molecules with the 32P-labeled cap01 structure (A) were incubated with enzymes as indicated in the presence of SAM. Purified product RNA was digested with RNase T2. Digestion products were resolved on 21% polyacrylamide/8 M urea gel and visualized by autoradiography. BAP protein was used as negative control. RNA with 32P-labeled cap structure created with the TbMTr2 enzyme was used as a reference. Specificity: autoradiography of two-dimensional chromatograms of 5′-phosphate nucleosides on thin layer cellulose plates. [α-32P] ATP-labeled in vitro transcribed cap01-RNA-GA was incubated with SAM in the absence, (B) or presence (C) of the hMTr2 protein. Product RNA was purified, cleaved by nuclease P1 and the resulting nucleotides were analyzed as described (44). 5′-monophosphate ribonucleosides of G, A, U, C, Am and m6A were used as standards. Substrate requirements: In vitro transcribed RNA-GG molecules with 32P-labeled cap0 (D) or capG (E) structure were incubated with one of the indicated enzymes, added in a given order, in the presence of SAM. After every modification step RNA molecules were purified by phenol/chloroform extraction and ethanol precipitation. Final products were digested and analyzed as described in legend for panel A. Asterisks indicate positions of 32P-labeled phosphates.
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Figure 2: hMTr2 activity and substrate requirements. Methyltransferase activity: In vitro transcribed RNA-GG molecules with the 32P-labeled cap01 structure (A) were incubated with enzymes as indicated in the presence of SAM. Purified product RNA was digested with RNase T2. Digestion products were resolved on 21% polyacrylamide/8 M urea gel and visualized by autoradiography. BAP protein was used as negative control. RNA with 32P-labeled cap structure created with the TbMTr2 enzyme was used as a reference. Specificity: autoradiography of two-dimensional chromatograms of 5′-phosphate nucleosides on thin layer cellulose plates. [α-32P] ATP-labeled in vitro transcribed cap01-RNA-GA was incubated with SAM in the absence, (B) or presence (C) of the hMTr2 protein. Product RNA was purified, cleaved by nuclease P1 and the resulting nucleotides were analyzed as described (44). 5′-monophosphate ribonucleosides of G, A, U, C, Am and m6A were used as standards. Substrate requirements: In vitro transcribed RNA-GG molecules with 32P-labeled cap0 (D) or capG (E) structure were incubated with one of the indicated enzymes, added in a given order, in the presence of SAM. After every modification step RNA molecules were purified by phenol/chloroform extraction and ethanol precipitation. Final products were digested and analyzed as described in legend for panel A. Asterisks indicate positions of 32P-labeled phosphates.

Mentions: To test the cap2-MTase activity of our candidate hMTr2, in vitro transcribed RNA-GG was capped with VCE in the presence of [α-32P] GTP and modified with VMT to form a cap01 substrate (Supplementary Figure S1, Table S1). This cap01-RNA was subjected to methylation with the purified FLAG-tagged recombinant hMTr2 protein, followed by RNase T2 digestion to generate 3′-monophosphate nucleotides. Presence of the 2′-O-ribose methylation prevents hydrolysis by RNase T2 generating RNA fragments of longer length (44). Digestion products were resolved on a polyacrylamide gel, along with control samples generated by the trypanosome cap2-MTase TbMTr2 as a positive control, and immunopurified FLAG-tagged BAP protein as a negative control (Figure 2A). When cap01-RNA was treated with hMTr2, a radioactive RNA fragment that comigrated with a RNase T2-resistant fragment from a sample treated with TbMTr2 was detected (Figure 2A, lanes 2 and 3), albeit at lower efficiency, corresponding to the predicted cap012 product. No methyl incorporation was observed in a control reaction with a purified BAP protein (Figure 2A, lane 4), demonstrating that the cap2 MTase activity was specifically from overexpressed hMTr2. Analogous experiments performed with the RNA-AG substrate also yielded a methylation product (data not shown).Figure 2.


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)

hMTr2 activity and substrate requirements. Methyltransferase activity: In vitro transcribed RNA-GG molecules with the 32P-labeled cap01 structure (A) were incubated with enzymes as indicated in the presence of SAM. Purified product RNA was digested with RNase T2. Digestion products were resolved on 21% polyacrylamide/8 M urea gel and visualized by autoradiography. BAP protein was used as negative control. RNA with 32P-labeled cap structure created with the TbMTr2 enzyme was used as a reference. Specificity: autoradiography of two-dimensional chromatograms of 5′-phosphate nucleosides on thin layer cellulose plates. [α-32P] ATP-labeled in vitro transcribed cap01-RNA-GA was incubated with SAM in the absence, (B) or presence (C) of the hMTr2 protein. Product RNA was purified, cleaved by nuclease P1 and the resulting nucleotides were analyzed as described (44). 5′-monophosphate ribonucleosides of G, A, U, C, Am and m6A were used as standards. Substrate requirements: In vitro transcribed RNA-GG molecules with 32P-labeled cap0 (D) or capG (E) structure were incubated with one of the indicated enzymes, added in a given order, in the presence of SAM. After every modification step RNA molecules were purified by phenol/chloroform extraction and ethanol precipitation. Final products were digested and analyzed as described in legend for panel A. Asterisks indicate positions of 32P-labeled phosphates.
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Related In: Results  -  Collection

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Figure 2: hMTr2 activity and substrate requirements. Methyltransferase activity: In vitro transcribed RNA-GG molecules with the 32P-labeled cap01 structure (A) were incubated with enzymes as indicated in the presence of SAM. Purified product RNA was digested with RNase T2. Digestion products were resolved on 21% polyacrylamide/8 M urea gel and visualized by autoradiography. BAP protein was used as negative control. RNA with 32P-labeled cap structure created with the TbMTr2 enzyme was used as a reference. Specificity: autoradiography of two-dimensional chromatograms of 5′-phosphate nucleosides on thin layer cellulose plates. [α-32P] ATP-labeled in vitro transcribed cap01-RNA-GA was incubated with SAM in the absence, (B) or presence (C) of the hMTr2 protein. Product RNA was purified, cleaved by nuclease P1 and the resulting nucleotides were analyzed as described (44). 5′-monophosphate ribonucleosides of G, A, U, C, Am and m6A were used as standards. Substrate requirements: In vitro transcribed RNA-GG molecules with 32P-labeled cap0 (D) or capG (E) structure were incubated with one of the indicated enzymes, added in a given order, in the presence of SAM. After every modification step RNA molecules were purified by phenol/chloroform extraction and ethanol precipitation. Final products were digested and analyzed as described in legend for panel A. Asterisks indicate positions of 32P-labeled phosphates.
Mentions: To test the cap2-MTase activity of our candidate hMTr2, in vitro transcribed RNA-GG was capped with VCE in the presence of [α-32P] GTP and modified with VMT to form a cap01 substrate (Supplementary Figure S1, Table S1). This cap01-RNA was subjected to methylation with the purified FLAG-tagged recombinant hMTr2 protein, followed by RNase T2 digestion to generate 3′-monophosphate nucleotides. Presence of the 2′-O-ribose methylation prevents hydrolysis by RNase T2 generating RNA fragments of longer length (44). Digestion products were resolved on a polyacrylamide gel, along with control samples generated by the trypanosome cap2-MTase TbMTr2 as a positive control, and immunopurified FLAG-tagged BAP protein as a negative control (Figure 2A). When cap01-RNA was treated with hMTr2, a radioactive RNA fragment that comigrated with a RNase T2-resistant fragment from a sample treated with TbMTr2 was detected (Figure 2A, lanes 2 and 3), albeit at lower efficiency, corresponding to the predicted cap012 product. No methyl incorporation was observed in a control reaction with a purified BAP protein (Figure 2A, lane 4), demonstrating that the cap2 MTase activity was specifically from overexpressed hMTr2. Analogous experiments performed with the RNA-AG substrate also yielded a methylation product (data not shown).Figure 2.

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
Related in: MedlinePlus