Efficient translation of poly(A)-deficient mRNAs in Saccharomyces cerevisiae.
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.
Proweller
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Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, New York 14642.
Butler
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D001483:Base Sequence; D017931:DNA Primers; D015870:Gene Expression; D005800:Genes, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D000073718:Pancreatitis-Associated Proteins; D016133:Polymerase Chain Reaction; D011062:Polynucleotide Adenylyltransferase / Q000037:antagonists & inhibitors; D011132:Polyribosomes / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014158:Transcription, Genetic
D017931:DNA Primers; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D012333:RNA, Messenger; D011062:Polynucleotide Adenylyltransferase