Related Experiment Videos
N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA
S M Carroll1, P Narayan, F M Rottman
1Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio 44106.
Abstract:
N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modification arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus far, m6A has been mapped to specific locations in eucaryotic mRNA and viral genomic RNA. We have now examined an intron-specific sequence of a modified bovine prolactin precursor RNA for the presence of this methylated nucleotide by using both transfected-cell systems and a cell-free system capable of methylating mRNA transcripts in vitro. The results indicate the final intron-specific sequence (intron D) of a prolactin RNA molecule does indeed possess m6A residues. When mapped to specific T1 oligonucleotides, the predominant site of methylation was found to be within the consensus sequence AGm6ACU. The level of m6A at this site is nonstoichiometric; approximately 24% of the molecules are modified in vivo. Methylation was detected at markedly reduced levels at other consensus sites within the intron but not in T1 oligonucleotides which do not contain either AAC or GAC consensus sequences. In an attempt to correlate mRNA methylation with processing, stably transfected CHO cells expressing augmented levels of bovine prolactin were treated with neplanocin A, an inhibitor of methylation. Under these conditions, the relative steady-state levels of the intron-containing nuclear precursor increased four to six times that found in control cells.
Insights
N6-methyladenosine (m6A) is a crucial RNA modification found in bovine prolactin precursor RNA introns. This study identifies specific methylation sites and shows m6A impacts RNA processing and intron retention.
Area of Science:
- Molecular Biology
- RNA Biology
- Epigenetics
Background:
- N6-methyladenosine (m6A) is a prevalent RNA modification in eukaryotic and viral RNAs.
- m6A occurs in mRNA and heterogeneous nuclear RNA, influencing gene expression.
- The canonical m6A consensus sequence is G/AAC.
Purpose of the Study:
- To investigate the presence and location of m6A in bovine prolactin precursor RNA introns.
- To determine the specific consensus sequence for m6A methylation within the intron.
- To explore the role of m6A in RNA processing and intron retention.
Main Methods:
- Utilized transfected-cell systems and cell-free systems for RNA methylation analysis.
- Mapped m6A sites using T1 oligonucleotide analysis.
- Employed neplanocin A, a methylation inhibitor, to study the effects on RNA processing.
Main Results:
- m6A residues were detected in the intron D sequence of bovine prolactin precursor RNA.
- The predominant methylation site was within the AGm6ACU consensus sequence.
- Approximately 24% of molecules showed in vivo m6A modification at the primary site.
- Inhibition of methylation led to a 4-6 fold increase in intron-containing precursor RNA levels.
Conclusions:
- The bovine prolactin precursor RNA intron D is a site for m6A modification.
- m6A methylation occurs at specific consensus sequences within the intron.
- m6A modification plays a role in regulating RNA processing and potentially influences intron retention.