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Locations of several novel 2'-O-methylated nucleotides in human 28S rRNA.

Rebane A, Roomere H, Metspalu A - BMC Mol. Biol. (2002)

Bottom Line: For nine of these snoRNAs, the respective ribose methylations in human 28S rRNA have been only presumptive.Seven novel ribose 2'-O-methylated residues (Am389, Am391, Gm1604, Gm1739, Gm2853, Cm3810, Gm4156, predicted by snoRNAs U26, U81, U80, U73, U50, U74 and U31, respectively) have been localized in human 28S rRNA.The total number of 2'-O-methylations in human rRNA is not yet known.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular and Cell Biology, Tartu University, Estonian Biocentre, 23 Riia St, Tartu 51010, Estonia. ana.rebane@yale.edu

ABSTRACT

Background: Ribose 2'-O-methylation, the most common nucleotide modification in mammalian rRNA, is directed by the C/D box small nucleolar RNAs (snoRNAs). Thus far, more than fifty putative human rRNA methylation guide snoRNAs have been identified. For nine of these snoRNAs, the respective ribose methylations in human 28S rRNA have been only presumptive.

Results: In this study, the methylation state of human 28S rRNA in the positions predicted by the snoRNAs U21, U26, U31, U48, U50, U73, U74, U80 and U81 was assessed using reverse transcription-based methods and several novel 2'-O-methylations were localized.

Conclusions: Seven novel ribose 2'-O-methylated residues (Am389, Am391, Gm1604, Gm1739, Gm2853, Cm3810, Gm4156, predicted by snoRNAs U26, U81, U80, U73, U50, U74 and U31, respectively) have been localized in human 28S rRNA. The total number of 2'-O-methylations in human rRNA is not yet known.

No MeSH data available.


Location of novel 2'-O-methylated nucleotides in human 28S rRNA using reverse transcription of partially hydrolyzed rRNA and reverse transcription of rRNA at low dNTP concentrations. The positions of novel 2'-O-methylated nucleotides are shown in bold together with the putative snoRNA guide in parenthesis. Three previously detected methylations Cm2838, Am3794 and Am3799 are also indicated. Lanes 0', 30', 45' and 60' show primer extension products of partially hydrolyzed 28S rRNA, where the hydrolysis time is 0, 30, 45 and 60 min, respectively. Lanes, labeled 1 mM, 0.02 mM and 0.002 mM represent reverse transcription products of 28S rRNA at the indicated dNTP concentrations. Lanes A, C, G and T contain dideoxy sequencing ladders. Note that primer extension products generated with 5'-end-phosphorylated oligodeoxynucleotide primers migrate about 1 nucleotide farther than the respective sequencing reaction products produced with oligodeoxynucleotide primers bearing a hydroxyl group at the 5' end and terminating with dideoxynucleotides.
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Figure 1: Location of novel 2'-O-methylated nucleotides in human 28S rRNA using reverse transcription of partially hydrolyzed rRNA and reverse transcription of rRNA at low dNTP concentrations. The positions of novel 2'-O-methylated nucleotides are shown in bold together with the putative snoRNA guide in parenthesis. Three previously detected methylations Cm2838, Am3794 and Am3799 are also indicated. Lanes 0', 30', 45' and 60' show primer extension products of partially hydrolyzed 28S rRNA, where the hydrolysis time is 0, 30, 45 and 60 min, respectively. Lanes, labeled 1 mM, 0.02 mM and 0.002 mM represent reverse transcription products of 28S rRNA at the indicated dNTP concentrations. Lanes A, C, G and T contain dideoxy sequencing ladders. Note that primer extension products generated with 5'-end-phosphorylated oligodeoxynucleotide primers migrate about 1 nucleotide farther than the respective sequencing reaction products produced with oligodeoxynucleotide primers bearing a hydroxyl group at the 5' end and terminating with dideoxynucleotides.

Mentions: Using reverse transcription of partially hydrolyzed RNA, we were able to identify five previously undetected 2'-O-methylated residues in the human 28S rRNA: Am389, Am391, Gm1604, Gm1739 and Gm4156, predicted by snoRNAs U26, U81, U80, U73 and U31, respectively (Fig. 1A, 1B, 1C and 1D). Interestingly, Gm4156 is most probably one of the three universal ribose methylations (corresponding to Gm2251 in E. Coli[4]) that are conserved among prokaryotic and eukaryotic rRNA. With reverse transcription at low dNTP concentrations, the presence of a methyl group at Cm3810 (predicted by U74) was observed (Fig. 1E). Before, 2'-O-methylation of either Cm3810 or Cm3811 had been suggested [1]. One previously undetected 2'-O-methylation, Gm2853 (predicted by U50), was identified using both methods (Fig. 1F). In addition, our primer extension experiments confirmed the presence of three earlier determined [1] 2'-O-methylations: Cm2838, Am3794 and Am3799 (Fig. 1E and 1F) that can be also viewed as "internal markers" of our experiments. Modification Am3799 have proposed to be directed by U79 [5], the snoRNA guides for modifications Cm2838 and Am3794 are not known.


Locations of several novel 2'-O-methylated nucleotides in human 28S rRNA.

Rebane A, Roomere H, Metspalu A - BMC Mol. Biol. (2002)

Location of novel 2'-O-methylated nucleotides in human 28S rRNA using reverse transcription of partially hydrolyzed rRNA and reverse transcription of rRNA at low dNTP concentrations. The positions of novel 2'-O-methylated nucleotides are shown in bold together with the putative snoRNA guide in parenthesis. Three previously detected methylations Cm2838, Am3794 and Am3799 are also indicated. Lanes 0', 30', 45' and 60' show primer extension products of partially hydrolyzed 28S rRNA, where the hydrolysis time is 0, 30, 45 and 60 min, respectively. Lanes, labeled 1 mM, 0.02 mM and 0.002 mM represent reverse transcription products of 28S rRNA at the indicated dNTP concentrations. Lanes A, C, G and T contain dideoxy sequencing ladders. Note that primer extension products generated with 5'-end-phosphorylated oligodeoxynucleotide primers migrate about 1 nucleotide farther than the respective sequencing reaction products produced with oligodeoxynucleotide primers bearing a hydroxyl group at the 5' end and terminating with dideoxynucleotides.
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Related In: Results  -  Collection

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Figure 1: Location of novel 2'-O-methylated nucleotides in human 28S rRNA using reverse transcription of partially hydrolyzed rRNA and reverse transcription of rRNA at low dNTP concentrations. The positions of novel 2'-O-methylated nucleotides are shown in bold together with the putative snoRNA guide in parenthesis. Three previously detected methylations Cm2838, Am3794 and Am3799 are also indicated. Lanes 0', 30', 45' and 60' show primer extension products of partially hydrolyzed 28S rRNA, where the hydrolysis time is 0, 30, 45 and 60 min, respectively. Lanes, labeled 1 mM, 0.02 mM and 0.002 mM represent reverse transcription products of 28S rRNA at the indicated dNTP concentrations. Lanes A, C, G and T contain dideoxy sequencing ladders. Note that primer extension products generated with 5'-end-phosphorylated oligodeoxynucleotide primers migrate about 1 nucleotide farther than the respective sequencing reaction products produced with oligodeoxynucleotide primers bearing a hydroxyl group at the 5' end and terminating with dideoxynucleotides.
Mentions: Using reverse transcription of partially hydrolyzed RNA, we were able to identify five previously undetected 2'-O-methylated residues in the human 28S rRNA: Am389, Am391, Gm1604, Gm1739 and Gm4156, predicted by snoRNAs U26, U81, U80, U73 and U31, respectively (Fig. 1A, 1B, 1C and 1D). Interestingly, Gm4156 is most probably one of the three universal ribose methylations (corresponding to Gm2251 in E. Coli[4]) that are conserved among prokaryotic and eukaryotic rRNA. With reverse transcription at low dNTP concentrations, the presence of a methyl group at Cm3810 (predicted by U74) was observed (Fig. 1E). Before, 2'-O-methylation of either Cm3810 or Cm3811 had been suggested [1]. One previously undetected 2'-O-methylation, Gm2853 (predicted by U50), was identified using both methods (Fig. 1F). In addition, our primer extension experiments confirmed the presence of three earlier determined [1] 2'-O-methylations: Cm2838, Am3794 and Am3799 (Fig. 1E and 1F) that can be also viewed as "internal markers" of our experiments. Modification Am3799 have proposed to be directed by U79 [5], the snoRNA guides for modifications Cm2838 and Am3794 are not known.

Bottom Line: For nine of these snoRNAs, the respective ribose methylations in human 28S rRNA have been only presumptive.Seven novel ribose 2'-O-methylated residues (Am389, Am391, Gm1604, Gm1739, Gm2853, Cm3810, Gm4156, predicted by snoRNAs U26, U81, U80, U73, U50, U74 and U31, respectively) have been localized in human 28S rRNA.The total number of 2'-O-methylations in human rRNA is not yet known.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular and Cell Biology, Tartu University, Estonian Biocentre, 23 Riia St, Tartu 51010, Estonia. ana.rebane@yale.edu

ABSTRACT

Background: Ribose 2'-O-methylation, the most common nucleotide modification in mammalian rRNA, is directed by the C/D box small nucleolar RNAs (snoRNAs). Thus far, more than fifty putative human rRNA methylation guide snoRNAs have been identified. For nine of these snoRNAs, the respective ribose methylations in human 28S rRNA have been only presumptive.

Results: In this study, the methylation state of human 28S rRNA in the positions predicted by the snoRNAs U21, U26, U31, U48, U50, U73, U74, U80 and U81 was assessed using reverse transcription-based methods and several novel 2'-O-methylations were localized.

Conclusions: Seven novel ribose 2'-O-methylated residues (Am389, Am391, Gm1604, Gm1739, Gm2853, Cm3810, Gm4156, predicted by snoRNAs U26, U81, U80, U73, U50, U74 and U31, respectively) have been localized in human 28S rRNA. The total number of 2'-O-methylations in human rRNA is not yet known.

No MeSH data available.