Redundant 3' end-forming signals for the yeast CYC1 mRNA.

Abstract

The cycl-512 mutation is a 38-bp deletion in the 3' untranslated region of the CYCI gene, which encodes iso-l-cytochrome c in Saccharomyces cerevisiae. This deletion caused a 90% reduction in the levels of the CYCI mRNA and protein because of the absence of the normal 3' end-forming signal. Although the 3' end-forming signal was not defined by previous The proper formation of mRNA 3' ends and transcription termination are essential for the optimal expression of genes in eukaryotes. Point mutations in the mRNA 3' end-forming signal, or a -deletion of the signal, can lead to drastic decreases in gene expression (1-4). Such a case was found in the yeast cycl-512 mutant (5). In addition to displaying a 90% decrease in CYC1+ mRNA and iso-1-cytochrome c, the cycl-512 mutation also caused the formation of aberrantly long transcripts with many discrete 3' termini ranging from the wild-type poly(A) site to endpoints >2000 nt downstream (5-9). Butler and Platt (10) found that the same 38-bp region deleted in cycl-512 was necessary for the proper cleavage of extended CYCI transcripts in vitro. In addition, the same 38-bp region was found by Russo and Sherman (11) to be able to cause the termination of transcription in vivo. Although this cycl-512 mutation and its properties were uncovered >10 years ago (5), and although this cycl-512 mutation was instrumental for defining 3' end-forming signals by examining sequences that restored the levels of the CYCI mRNAs (6), the exact 3' end-forming signals of the wild-type CYC1 gene were not previously determined. Because of the inability to inactivate the 3' end-forming signal by nested deletions and multiple base-pair substitution within the 38-bp region, workers suggested that the 3' end-forming signal was only an A+T-rich region (7) or a symmetrical RNA structure (8). Russo et al (9) suggested that the CYC1 3' end-forming signals may have functional redundancy, whereas Wu (12) suggested three TA pairs may be important.Signals required for 3' end formation were uncovered by examining intragenic revertants and oligonucleotide-directed alterations that restored the cycl-512 defect (6,9

3′-end-forming signals of yeast mRNA

“…Deletion or base pair substitution analysis of AAUAAA showed that it was not absolutely required in the ADE8 and ADH2 genes (17,19). These observations, along with our results in this study, are reminiscent of the study of the CYC1 efficiency elements in which we found that none of the 10-bp deletions within the 38-bp region can completely abolish CYC1 mRNA 3Ј end formation because of the redundancy of the signal (13,31).…”

Coupled RNA polymerase II transcription and 3′ end formation with yeast whole-cell extracts

“…3D, lane 1). DNA sequencing of 12 independent 39 ends revealed that 39 end cleavage in vitro occurred in 11 cases between the CYA at position 504 relative to the CYC1 translational start codon and thus at the same position as in cells (Guo et al 1995). All 39 ends also carried poly(A) stretches that varied in length between 16 and 68 adenosine residues, showing that polyadenylation and poly(A) length control occurred efficiently in our in vitro system (see also Fig.…”

Protein factors in pre-mRNA 3′-end processing

“…The sequence UAUAUA provided the greatest effect on 3′-end processing with the U at the first and fifth positions being the most critical for function [65]. A large-scale analysis of the 3′ untranslated region of 1017 yeast nuclear transcripts showed that more than half of them (52%) contained the optimal EE sequence (UAUAUA) [66]. While the EE improves the efficiency, it is not required for cleavage.…”