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Efficient translation of poly(A)-deficient mRNAs in Saccharomyces cerevisiae
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, New York 14642.
Abstract:
The polyadenylate tail of eukaryotic mRNAs is thought to influence various metabolic phenomena including mRNA stability, translation initiation, and nucleo-cytoplasmic transport. We have analyzed the fate of mRNAs following inactivation of poly(A) polymerase in Saccharomyces cerevisiae containing a temperature-sensitive, lethal mutation (pap1-1) in the gene for poly(A) polymerase (PAP1). Inactivation of poly(A) polymerase (Pap1) by shifting cells to the nonpermissive temperature resulted in the loss of at least 80% of measurable poly(A) within 60 min. Northern blot analysis revealed the disappearance of some mRNAs (CYH2 and HIS4) consistent with a role for poly(A) tails in mRNA stability. However, other mRNAs (TCM1, PAB1, ACT1, and HTB2) accumulate as poly(A)-deficient (A < approximately 25) transcripts as defined by an inability to bind oligo(dT)-cellulose. Sucrose density gradient analysis of polyribosomes revealed a twofold reduction in the amount of each size class of polyribosomes in shifted cells and a commensurate increase in free ribosomes. However, poly(A)-deficient mRNAs in shifted cells remain associated with the same size polyribosomes as poly(A)+ mRNAs in unshifted cells, indicating normal initiation of translation. RNase mapping of transcripts from pap1-1 cells revealed PAB1 mRNA to be poly(A)- whereas TCM1 exists as equal amounts of poly(A)- and poly(A)+ mRNA 60 min after shift. Interestingly, both of these classes of TCM1 mRNA appear in similar amounts in each polyribosome fraction indicating that ribosomes may not distinguish between them. These findings suggest that under conditions of excess translational capacity, poly(A)- and poly(A)+ mRNAs may initiate translation with comparable efficiencies.
Insights
Polyadenylate polymerase inactivation in yeast affects mRNA stability and translation. Poly(A)-deficient mRNAs can still initiate translation, suggesting the tail
Area of Science:
- Molecular Biology
- Yeast Genetics
- mRNA Metabolism
Background:
- The polyadenylate tail of eukaryotic mRNAs is crucial for mRNA stability, translation, and transport.
- Poly(A) polymerase (PAP) synthesizes these tails.
- Understanding PAP's role requires studying mRNA fate upon its inactivation.
Purpose of the Study:
- To investigate the impact of poly(A) polymerase inactivation on mRNA stability and translation initiation in Saccharomyces cerevisiae.
- To determine which mRNAs are affected by the loss of polyadenylation.
- To assess the translational competence of poly(A)-deficient mRNAs.
Main Methods:
- Utilized a temperature-sensitive mutant (pap1-1) of Saccharomyces cerevisiae.
- Inactivated poly(A) polymerase (Pap1) by shifting cells to a nonpermissive temperature.
- Analyzed mRNA fate using Northern blot, oligo(dT)-cellulose binding, sucrose density gradient centrifugation, and RNase mapping.
Main Results:
- Poly(A) polymerase inactivation led to a rapid loss of poly(A) tails (>80% within 60 min).
- Some mRNAs (CYH2, HIS4) degraded, indicating a role for poly(A) in stability.
- Other mRNAs (TCM1, PAB1, ACT1, HTB2) accumulated as poly(A)-deficient transcripts.
- Poly(A)-deficient mRNAs remained associated with polyribosomes, suggesting normal translation initiation.
- Ribosomes appeared to translate both poly(A)+ and poly(A)- TCM1 mRNA efficiently.
Conclusions:
- Polyadenylation is essential for the stability of certain mRNAs but not all.
- Poly(A)-deficient mRNAs can initiate translation, challenging the universal requirement of poly(A) tails for this process.
- Under conditions of ample translational machinery, poly(A)+ and poly(A)- mRNAs exhibit similar translation initiation efficiencies.