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Methylated nucleosides in globin mRNA from mouse nucleated erythroid cells
Abstract:
Poly(A)-containing mRNAs labeled with [methyl-3H]methionine were isolated from nucleated erythroid cells obtained from the spleens of anemic mice. The RNAs were further separated into non-globin poly(A)-containing RNAs and highly purified globin mRNA by globin cDNA-cellulose affinity chromatography. DEAE-Sephadex column chromatography of the T2 ribonuclease digestion products of the cDNA-purified globin mRNA fraction yielded methylated resistant fragments with charges of -4.7 (Cap 1) and -5.3 (Cap 2). Digestion of the non-globin RNA fraction revealed a similar pattern with the addition of a methylated mononucleotide identified as 6-methyladenosine at -2 charges. Alkaline phosphatase treatment of the T2 resistant fragments reduced their charges by approximately 2, which is consistent with the removal of one terminal phosphate. Treatment of the globin T2 and alkaline phosphatase-resistant fragments withpenicillium P1 nuclease and alkaline phosphatase yielded a P1-resistant core structure in both fragments. In addition to the core, 2'-O-methylcytidine (Cm) was released from the more negatively charged globin fragment. The P1-resistant cores of the cap structures eluted from DEAE-Sephadex with the known standard m2G5'ppp5'Am and were found to be pyrophosphatase-sensitive establishing a 5'-5'-triphosphate linkage. The pyrophosphatase and alkaline phosphatase digestion products of the globin Cap 1 and Cap 2 core structures were analyzed by high voltage electrophoresis and paper chromatography and found to be 7-methyiguanosine (m7G) and the dimethylated nucleoside 6-methyl-2'-O-methyladenosine (N6mAm). A small amount of the singularly methylated adenosine, 2'-O-methyladenosine (Am) was also observed. The predominant sequences of the methylated nucleosides in the globin cap structures are therefore m7G5'ppp5'N6mAm and m7G5'ppp5'N6mAmpCm.
Insights
This study identifies methylated nucleoside sequences in mouse erythroid cell mRNA caps, revealing m7G5'ppp5'N6mAm and m7G5'ppp5'N6mAmpCm as predominant structures. These findings enhance understanding of mRNA processing and regulation.
Area of Science:
- Molecular Biology
- Biochemistry
- RNA Biology
Background:
- Messenger RNAs (mRNAs) possess 5' cap structures crucial for stability and translation.
- Methylated nucleosides within these cap structures play vital roles in gene expression regulation.
Purpose of the Study:
- To elucidate the precise methylated nucleoside sequences of cap structures in poly(A)-containing mRNAs from mouse erythroid cells.
- To characterize the cap structures of both globin and non-globin mRNAs.
Main Methods:
- Isolation and purification of poly(A)-containing mRNAs from anemic mouse spleen erythroid cells.
- Enzymatic digestion (T2 ribonuclease, P1 nuclease, alkaline phosphatase, pyrophosphatase) and chromatographic separation (DEAE-Sephadex, globin cDNA-cellulose).
- Analysis of digestion products using high voltage electrophoresis and paper chromatography.
Main Results:
- Identified methylated resistant fragments (Cap 1 and Cap 2) in globin mRNA, and similar patterns with 6-methyladenosine in non-globin mRNA.
- Determined the presence of a 5'-5'-triphosphate linkage in the cap structures.
- Characterized the predominant cap sequences as m7G5'ppp5'N6mAm and m7G5'ppp5'N6mAmpCm, with minor amounts of Am.
Conclusions:
- Established the detailed primary sequences of methylated cap structures in mouse erythroid mRNA.
- Provided insights into the heterogeneity of mRNA cap methylation in erythroid cells.
- Contributed to understanding the structural diversity of mRNA 5' caps and their potential regulatory functions.