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Ribosome concentration contributes to discrimination against poly(A)- mRNA during translation initiation in
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Abstract:
Inactivation of Saccharomyces cerevisiae poly(A) polymerase in a strain bearing the temperature-sensitive lethal pap1-1 mutation results in the synthesis of poly(A)- mRNAs that initiate translation with surprising efficiency. Translation of poly(A)- mRNAs after polyadenylation shut-off might result from an increase in the ratio of ribosomes and associated translation factors to mRNA, caused by the inability of poly(A)- mRNAs to accumulate to normal levels. To test this hypothesis, we used ribosomal subunit protein gene mutations to decrease either 40 or 60 S ribosomal subunit concentrations in strains carrying the pap1-1 mutation. Polyadenylation shut-off in such cells results in a nearly normal ratio of ribosomes to mRNA as revealed by polyribosome sedimentation analysis. Ribonuclease protection and Northern blot analyses showed that a significant percentage of poly(A)-deficient and poly(A)- mRNA associate with smaller polyribosomes compared with cells with normal ribosome levels. Analysis of the ratio of poly(A)-deficient and poly(A)- forms of a specific mRNA showed relatively more poly(A)- mRNA sedimenting with 20-60 S complexes than do poly(A)+ forms, suggesting a block in an early step of the translation initiation of the poly(A)- transcripts. These findings support models featuring the poly(A) tail as an enhancer of translation and suggest that the full effect of a poly(A) tail on the initiation strength of a mRNA may require competition for a limited number of free ribosomes or translation factors.
Insights
Polyadenylation (PAP) is crucial for efficient mRNA translation. Even without PAP, yeast mRNA initiates translation effectively, suggesting PAP enhances translation initiation by competing for ribosomes and translation factors.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Gene Expression Regulation
Background:
- The poly(A) tail of messenger RNA (mRNA) is generally considered essential for efficient translation initiation.
- In Saccharomyces cerevisiae, the pap1-1 mutation inactivates poly(A) polymerase, leading to poly(A)-deficient mRNAs.
Purpose of the Study:
- To investigate the role of the poly(A) tail in mRNA translation initiation.
- To test the hypothesis that reduced mRNA levels increase the ribosome-to-mRNA ratio, enhancing translation of poly(A)-deficient mRNAs.
Main Methods:
- Utilized temperature-sensitive lethal pap1-1 yeast strains.
- Introduced mutations in ribosomal subunit protein genes to alter ribosome concentrations.
- Performed polyribosome sedimentation analysis, ribonuclease protection assays, and Northern blot analysis.
Main Results:
- Polyadenylation shut-off in pap1-1 cells with reduced ribosome levels maintained a near-normal ribosome-to-mRNA ratio.
- Poly(A)-deficient and poly(A)- mRNAs associated with smaller polyribosomes compared to controls.
- Poly(A)- mRNAs showed a greater association with 20-60S complexes, indicating a translation initiation block.
Conclusions:
- The poly(A) tail enhances mRNA translation initiation strength.
- This enhancement may involve competition for limited ribosomes or translation factors.
- Findings support models where poly(A) tails act as translation enhancers.