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Evidence for a complex class of nonadenylated mRNA in Drosophila
Abstract:
The amount, by mass, of poly(A+) mRNA present in the polyribosomes of third-instar larvae of Drosophila melanogaster, and the relative contribution of the poly(A+) mRNA to the sequence complexity of total polysomal RNA, has been determined. Selective removal of poly(A+) mRNA from total polysomal RNA by use of either oligo-dT-cellulose, or poly(U)-sepharose affinity chromatography, revealed that only 0.15% of the mass of the polysomal RNA was present as poly(A+) mRNA. The present study shows that this RNA hybridized at saturation with 3.3% of the single-copy DNA in the Drosophila genome. After correction for asymmetric transcription and reactability of the DNA, 7.4% of the single-copy DNA in the Drosophila genome is represented in larval poly(A+) mRNA. This corresponds to 6.73 X 10(6) nucleotides of mRNA coding sequences, or approximately 5,384 diverse RNA sequences of average size 1,250 nucleotides. However, total polysomal RNA hybridizes at saturation to 10.9% of the single-copy DNA sequences. After correcting this value for asymmetric transcription and tracer DNA reactability, 24% of the single-copy DNA in Drosophila is represented in total polysomal RNA. This corresponds to 2.18 X 10(7) nucleotides of RNA coding sequences or 17,440 diverse RNA molecules of size 1,250 nucleotides. This value is 3.2 times greater than that boserved for poly(A+) mRNA, and indicates that congruent to 69% of the polysomal RNA sequence complexity is contributed by nonadenylated RNA. Futhermore, if the number of different structural genes represented in total polysomal RNA is congruent to 1.7 X 10(4), then the number of genes expressed in third-instar larvae exceeds the number of chromomeres in Drosophila by about a factor of three. This numbeology indicates that the number of chromomeres observed in polytene chromosomes does not reflect the number of structural gene sequences in the Drosophila genome.
Insights
Polyadenylated mRNA (poly(A+) mRNA) constitutes a small fraction of total polysomal RNA in Drosophila larvae. Most of the sequence complexity in larval polysomes comes from nonadenylated RNA, challenging the chromomere model of gene expression.
Area of Science:
- Molecular Biology
- Genomics
- Developmental Biology
Background:
- Polyadenylated mRNA (poly(A+) mRNA) plays crucial roles in eukaryotic gene expression and stability.
- Understanding the contribution of poly(A+) mRNA to the overall transcriptome is essential for deciphering gene regulation.
Purpose of the Study:
- To quantify the mass and sequence complexity of poly(A+) mRNA in Drosophila melanogaster third-instar larvae.
- To determine the proportion of the Drosophila genome represented by poly(A+) mRNA and nonadenylated RNA in polysomes.
Main Methods:
- Affinity chromatography using oligo-dT-cellulose or poly(U)-sepharose to isolate poly(A+) mRNA.
- RNA-DNA hybridization assays to determine sequence complexity and genome representation.
- Calculations to correct for asymmetric transcription and DNA reassociation kinetics.
Main Results:
- Poly(A+) mRNA comprises only 0.15% of the total mass of polysomal RNA.
- Poly(A+) mRNA represents 7.4% of the single-copy Drosophila genome, corresponding to approximately 5,384 diverse RNA sequences.
- Nonadenylated RNA contributes significantly to polysomal RNA complexity, accounting for approximately 69%.
Conclusions:
- The majority of sequence complexity in larval polysomal RNA is derived from nonadenylated RNA, not poly(A+) mRNA.
- The number of expressed genes in Drosophila larvae likely exceeds the number of observed chromomeres, suggesting limitations of the chromomere model.