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Methylated, blocked 5' termini of yeast mRNA
Abstract:
mRNAs of the yeast, Saccharomyces cerevisiae, have modified 5' termini similar but not identical with those present in the higher eukaryotes. Poly(A)-containing RNA isolated from spheroplasts or from polysomes of cells labeled with [methyl-3H]methoionine or 32Pi contain methylated, blocked 5'-terminal structures of composition: m7G(5')pppAp and m7G(5')pppGp, occurring in a relative distribution of 75% and 25%, respectively. Furthermore there is no N6-methyladenosine nor any other methylated nucleoside in the entire molecule of poly(A)-containing RNA. Experiments with a mutant, temperature-sensitive for the transport of mRNA to cytoplasm, suggest that the methylation and addition of the blocking group take place in the nucleus and that the genetic lesion in the mutant does not affect these mechanisms.
Insights
Yeast messenger RNAs (mRNAs) possess modified 5' termini, specifically methylated cap structures like m7G(5')pppAp and m7G(5')pppGp. These modifications occur in the nucleus before mRNA transport to the cytoplasm.
Area of Science:
- Molecular Biology
- Yeast Genetics
- RNA Biology
Background:
- Eukaryotic messenger RNAs (mRNAs) feature a 5' cap structure crucial for stability and translation.
- The specific nature of 5' cap modifications in the yeast Saccharomyces cerevisiae remained incompletely characterized compared to higher eukaryotes.
Purpose of the Study:
- To elucidate the precise chemical composition and structure of the 5' termini of poly(A)-containing RNA in Saccharomyces cerevisiae.
- To investigate the cellular location and genetic control of 5' cap methylation and addition in yeast.
Main Methods:
- Isolation of poly(A)-containing RNA from Saccharomyces cerevisiae spheroplasts and polysomes.
- Metabolic labeling of RNA with [methyl-3H]methionine and 32P.
- Analysis of 5'-terminal structures using chromatographic and enzymatic methods.
- Characterization of mRNA transport mutants.
Main Results:
- Identified two major 5'-terminal methylated structures: m7G(5 ransformers')pppAp (75%) and m7G(5 ransformers')pppGp (25%).
- Confirmed the absence of N6-methyladenosine and other methylated nucleosides in the poly(A)-containing RNA.
- Evidence from a temperature-sensitive mRNA transport mutant suggests cap methylation and blocking occur in the nucleus.
Conclusions:
- Yeast mRNA 5' termini are methylated and blocked, analogous to higher eukaryotes but with distinct compositions.
- Cap modification and addition are nuclear processes in yeast.
- The studied genetic lesion affecting mRNA transport does not impact these 5' end modifications.