Harper, J. E., Miceli, S. M., Roberts, R. J., Manley, J. L. (October 1990) Sequence Specificity of the Human Messenger-Rna N6-Adenosine Methylase Invitro. Nucleic Acids Research, 18 (19). pp. 5735-5741. ISSN 0305-1048
Abstract
N6-adenosine methylation is a frequent modification of mRNAs and their precursors, but little is known about the mechanism of the reaction or the function of the modification. To explore these questions, we developed conditions to examine N6-adenosine methylase activity in HeLa cell nuclear extracts. Transfer of the methyl group from S-[3H methyl]-adenosylmethionine to unlabeled random copolymer RNA substrates of varying ribonucleotide composition revealed a substrate specificity consistent with a previously deduced consensus sequence, Pu[G greater than A]AC[A/C/U]. 32-P labeled RNA substrates of defined sequence were used to examine the minimum sequence requirements for methylation. Each RNA was 20 nucleotides long, and contained either the core consensus sequence GGACU, or some variation of this sequence. RNAs containing GGACU, either in single or multiple copies, were good substrates for methylation, whereas RNAs containing single base substitutions within the GGACU sequence gave dramatically reduced methylation. These results demonstrate that the N6-adenosine methylase has a strict sequence specificity, and that there is no requirement for extended sequences or secondary structures for methylation. Recognition of this sequence does not require an RNA component, as micrococcal nuclease pretreatment of nuclear extracts actually increased methylation efficiency.
| Item Type: | Paper |
|---|---|
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA methylation bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > methyltransferase bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > mRNA |
| CSHL Authors: | |
| Communities: | CSHL labs > Roberts lab |
| Depositing User: | Matt Covey |
| Date: | 11 October 1990 |
| Date Deposited: | 08 Feb 2016 17:49 |
| Last Modified: | 08 Feb 2016 17:49 |
| PMCID: | PMC332308 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/32335 |
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