A small segment of the MAT alpha 1 transcript promotes mRNA decay in Saccharomyces cerevisiae: a stimulatory role for rare codons.

Abstract

Differences in decay rates of eukaryotic transcripts can be determined by discrete sequence elements within mRNAs. Through the analysis of chimeric transcripts and internal deletions, we have identified a 65-nucleotide segment of the AL4Tal mRNA coding region, termed the AL4TaJ instability element, that is sufficient to confer instability to a stable PGKI reporter transcript and that accelerates turnover of the unstable AL4TaI mRNA.This 65-nucleotide element is composed of two parts, one located within the 5' 33 nucleotides and the second located in the 3' 32 nucleotides. The first part, which can be functionally replaced by sequences containing rare codons, is unable to promote rapid decay by itself but can enhance the action of the 3' 32 nucleotides (positions 234 to 266 in the AL4Tal mRNA) in accelerating turnover. A second portion of the MA4Ta) mRNA (nucleotides 265 to 290) is also sufficient to destabilize the PGKI reporter transcript when positioned 3' of rare codons, suggesting that the 3' half of the MATal instability element is functionally reiterated within the MA4TcJ mRNA. The observation that rare codons are part of the 65-nucleotide MAToJ instability element suggests possible mechanisms through which translation and mRNA decay may be linked.The regulation of eukaryotic gene expression can be significantly affected by differences in mRNA decay rates. Critical to the understanding of mRNA turnover is a detailed knowledge of the mRNA features that specify differences in decay rates. Recent experiments, examining the decay rates of chimeric and/or mutant transcripts, suggest that at least in some cases, there are discrete sequence elements that affect mRNA turnover rates (for reviews, see references 19 and 28). These instability elements have been described in the protein coding and/or 3' untranslated regions of a small number of mRNAs.Determinants of mRNA stability have been identified in the protein coding regions of mammalian (14,32,41) and yeast (16,17,27,36) transcripts. These observations suggest that the coding region will be a common location for sequences that affect mRNA half-lives (t1j2s). However, the critical features of such coding-region stability determinants, and how these elements function in the presence of translocating ribosomes, are largely unknown. Recent results suggest that recognition of information in the coding region specifying mRNA decay rates may occur by two distinctly different mechanisms. In the case of the mammalian 3-tubulin mRNA, recognition occurs at the level of the nascent peptide, demonstrating how translation and decay can be linked (44). Alternatively, coding-region determinants in c-fos and c-myc mRNAs may be recognized by proteins that bind directly to the RNA (6, 10), although the relationship between protein binding, mRNA decay, and translocating ribosomes is unclear.The genetic approaches possible in the yeast Saccharomyces cerevisiae make this organism a useful system for the analysis of eukaryotic mRNA decay. Previously, we reported that a 363-nucleotide...

The Target of Rapamycin Signaling Pathway Regulates mRNA Turnover in the YeastSaccharomyces cerevisiae

“…Total RNA was isolated from frozen cell pellets, as previously described (Caponigro et al, 1993). Unless otherwise noted, RNA was fractionated on 1.75% formaldehyde agarose gels run for 3.5 h at 70 V, transferred to Zeta-Probe (Bio-Rad, Richmond, CA) with 10ϫ SSC, UV-crosslinked, and probed by standard methods.…”

“…Northern analysis was quantified with the use of a Storm860 Phosphorimager (Molecular Dynamics, Eugene, OR) and ImageQuant software (Molecular Dynamics). A RNA polymerase III transcript, 7S RNA, which was detected with oRP100, was used to correct for differences in RNA loading (Caponigro et al, 1993).…”

“…Northern analysis was quantified with the use of a Storm860 Phosphorimager (Molecular Dynamics, Eugene, OR) and ImageQuant software (Molecular Dynamics). A RNA polymerase III transcript, 7S RNA, which was detected with oRP100, was used to correct for differences in RNA loading (Caponigro et al, 1993).mRNA Half-life Analysis. mRNA decay rates were determined by thermal inactivation of rpb1-1 with the use of a protocol modified from Herrick et al (1990).…”

Identification and Characterization of a Sequence Motif Involved in Nonsense-Mediated mRNA Decay

“…Our results, as well as work from other laboratories, strongly indicate that the processes of mRNA turnover and translation are intimately linked and that understanding of this relationship is critical in elucidating the mechanism of mRNA decay (2, 5, 9, 11, 15, 21, 23, 29-31, 41, 53, 55-59, 76). The role of translation in determination of mRNA decay rates is direct, and, for certain instability elements identified in protein coding regions and 3Ј untranslated regions (3Ј-UTRs), translation of the protein coding region must occur in order for them to function (2,11,15,21,23,29,41,53,60,66,76).…”

“…Our results, as well as work from other laboratories, strongly indicate that the processes of mRNA turnover and translation are intimately linked and that understanding of this relationship is critical in elucidating the mechanism of mRNA decay (2, 5, 9, 11, 15, 21, 23, 29-31, 41, 53, 55-59, 76). The role of translation in determination of mRNA decay rates is direct, and, for certain instability elements identified in protein coding regions and 3Ј untranslated regions (3Ј-UTRs), translation of the protein coding region must occur in order for them to function (2,11,15,21,23,29,41,53,60,66,76).One clear example of the relationship between translation and mRNA decay is the observation that nonsense mutations in a gene can reduce the abundance of the mRNA transcribed from that gene in a process we term nonsense-mediated mRNA decay (55,56,58). Reduced mRNA levels or decreased stabilities of nonsense-containing transcripts have been observed in both prokaryotes and eukaryotes (6-8, 13, 14, 18, 20, 22, 42, 44-47, 51, 54, 56, 71, 75).…”