A cell-free extract from yeast cells for studying mRNA turnover

Vreken, P.; Buddelmeijer, Nienke; Raué, Hendrik A.

doi:10.1093/nar/20.10.2503

Cited by 19 publications

(6 citation statements)

References 41 publications

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“…The fact that the 5' ends of the downstream cleavage products identified in this study map to the 3'-terminal G, or in one case the penultimate U, residue of a 5'-GGUG-3' sequence indicates a certain measure of sequence specificity as has been noted also in the case of apolipoprotein II mRNA (6). Mutational analysis of the target region by means of an in vitro system from S. cerevisiae 20B-12 cells that mimics the in vivo endonucleolytic cleavage events supports this idea, since it showed that changing GGUG to GGUA abolished 5'-end formation at that particular sequence (61). However, it is also clear that more than just a GGUG motif is required, since the target region contains additional copies of this motif at which 5' ends were not detected by primer extension analysis (Fig.…”

Section: Discussionmentioning

confidence: 84%

The rate-limiting step in yeast PGK1 mRNA degradation is an endonucleolytic cleavage in the 3'-terminal part of the coding region.

Vreken¹,

Raué²

1992

Mol. Cell. Biol.

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Insertion of an 18-nucleotide-long poly(G) tract into the 3'-terminal untranslated region of yeast phosphoglycerate kinase (PGKI) mRNA increases its chemical half-life by about a factor of 2 (P. Vreken, R. Van der Veen, V. C. H. F. de Regt, A. L. de Maat, R. J. Planta, and H. A. Raue, Biochimie 73:729-737, 1991). In this report, we show that this insertion also causes the accumulation of a degradation intermediate extending from the poly(G) sequence down to the transcription termination site. Reverse transcription and Si nuclease mapping experiments demonstrated that this intermediate is the product of shorter-lived primary fragments resulting from endonucleolytic cleavage immediately downstream from the U residue of either of two 5'-GGUG-3' sequences present between positions 1100 and 1200 close to the 3' terminus (position 1251) of the coding sequence. Similar endonucleolytic cleavages appear to initiate degradation of wild-type PGKI mRNA. Insertion of a poly(G) tract just upstream from the AUG start codon resulted in the accumulation of a 5'-terminal degradation intermediate extending from the insertion to the 1100-1200 region. RNase H degradation in the presence of oligo(dT) demonstrated that the wild-type and mutant PGKI mRNAs are deadenylated prior to endonucleolytic cleavage and that the half-life of the poly(A) tail is three-to sixfold lower than that of the remainder of the mRNA. Thus, the endonucleolytic cleavage constitutes the rate-limiting step in degradation of both wild-type and mutant PGKI transcripts, and the resulting fragments are degraded by a 5'-*3' exonuclease, which appears to be severely retarded by a poly(G) sequence.Over the past decade, it has become evident that the pattern of gene expression in a cell is determined not only by factors controlling transcription but to a large extent also by posttranscriptional events, including the rate of degradation of the individual mRNAs. In both prokaryotic and eukaryotic cells, different mRNAs show sometimes considerable differences in stability that are of fundamental importance in establishing their relative cellular levels. Furthermore, there are numerous examples of adjustments in the half-life of particular mRNAs in response to specific stimuli that play a major role in the adaptation of the cells to changing conditions (for recent reviews, see references 1, 4, 9, 12, and 48).To gain more insight into the manner in which turnover of their transcripts contributes to the control of expression of particular genes, considerable effort is being put into the characterization of structural features within mRNA governing the rate of decay, as well as the trans-acting factors involved. As a result, a number of structural determinants of prokaryotic as well as eukaryotic mRNA stability have now been identified. Among the eukaryotic stability determinants so far characterized are the AU-rich sequences responsible for the rapid turnover of a group of mRNAs encoding lymphokines, interferons, and cellular growth factors (13,46,49,50), the stem-loop struc...

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Section: Discussionmentioning

confidence: 84%

The rate-limiting step in yeast PGK1 mRNA degradation is an endonucleolytic cleavage in the 3'-terminal part of the coding region.

Vreken¹,

Raué²

1992

Mol. Cell. Biol.

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“…Vreken et al (49) found that insertion of a G18 tract into the 3' UTR of yeast PGKI mRNA increases its half-life by a factor of 2 without affecting its translation. Further studies of Vreken and Raue (48) and Vreken et al (47) demonstrated that the same insertion causes the accumulation of high levels of a short fragment of the mRNA extending from the G18 sequence to the mRNA cleavage-polyadenylation site. Primer extension and S1 nuclease mapping studies showed that the fragment results from an apparent 5'-*3' exoribonucleolytic hydrolysis of short-lived fragments formed by endonucleolytic cleavages close to the 3' terminus of the coding sequence of the PGKI mRNA.…”

Section: Discussionmentioning

confidence: 97%

Yeast cells lacking 5'-->3' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5' cap structure.

Hsu¹,

Stevens²

1993

Mol. Cell. Biol.

365

319

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Analysis of the slowed turnover rates of several specific mRNA species and the higher cellular levels of some of these mRNAs in Saccharomyces cerevisiae lacking 5'--3' exoribonuclease 1 (xrnl cells) has led to the finding that these yeast contain higher amounts of essentially full-length mRNAs that do not bind to oligo(dT)-cellulose. On the other hand, the length of mRNA poly(A) chains found after pulse-labeling of cells lacking the exoribonuclease, the cellular rate of synthesis of oligo(dT)-bound mRNA, and the initial rate of its deadenylation appeared quite similar to the same measurements in wild-type yeast cells. Examination of the 5' cap structure status of the poly(A)-deficient mRNAs by comparative analysis of the m7G content of poly(A)-and poly(A)+ RNA fractions of wild-type and xrnl ceUs suggested that the xrnl poly(A)-mRNA fraction is low in cap structure content. Further analysis of the 5' termini by measurements of the rate of 5'-3' exoribonuclease 1 hydrolysis of specific full-length mRNA species showed that approximately 50%o of the xrnl poly(A)-deficient mRNA species lack the cap structure. Primer extension analysis of the 5' terminus of ribosomal protein 51A (RPSIA) mRNA showed that about 30% of the poly(A)-deficient molecules of the xrnl cells are slightly shorter at the 5' end. The finding of some accumulation of poly(A)-deficient mRNA species partially lacking the cap structure together with the reduction of the rate of mRNA turnover in cells lacking the enzyme suggest a possible role for 5'--3' exoribonuclease 1 in the mRNA turnover process.5'-3' exoribonuclease 1 from Saccharomyces cerevisiae has been highly purified and characterized (40,44). In recent studies of gene cloning and sequencing (21,22), the enzyme was designated XRN1. The enzyme was found to be a 160-kDa protein acting as a processive exoribonuclease and producing 5'-mononucleotides by a 5'--3' direction of hydrolysis (40, 44). The mode of hydrolysis by XRN1 was studied in detail by using poly(A), rRNA, and oligo(A) as substrates. Capped mRNA was quite resistant to hydrolysis (39). Poly(I) was not hydrolyzed by the enzyme (44). As a step toward determining the metabolic role of XRN1, the yeast gene designated XRN1 was cloned and disrupted to determine the effect on yeast cell growth (22 protein's role in mRNA metabolism. The purification and characterization of a yeast mRNA-decapping enzyme yielding as products m7GDP and mRNA chains with 5'-phosphate termini led us to suggest that the two enzymes might be involved in mRNA turnover (41,42).In this report, we describe further analysis of mRNA species found in an XRN1 gene disruptant of S. cerevisiae. Special emphasis was placed on analyzing the structure of the 5' termini of the mRNAs to determine the status of the 5' cap structure. Essentially full-length mRNA species that are both poly(A) deficient and low in cap structure content are found in the xrnl yeast cells. The possibility that such mRNA molecules are intermediates in mRNA turnover is discussed. MATERIALS AND METHODSYea...

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“…This pathway has also been shown to be a primary pathway for PGK1 mRNA degradation (14,17); however, it has also been shown that the deadenylated PGK1 mRNA is degraded in a 3Ј-to-5Ј direction (17). Additionally, in vivo and in vitro evidence shows that following poly(A) tail removal, endonucleolytic cleavages occur near the 3Ј end of the PGK1 coding region, generating products that are rapidly degraded in a 5Ј-to-3Ј direction (38,39).…”

Section: G Cap(؊)mentioning

confidence: 99%

Degradation of the Soybean Ribulose-1,5-Bisphosphate Carboxylase Small-Subunit mRNA, SRS4, Initiates with Endonucleolytic Cleavage

Tanzer

Meagher

1995

Molecular and Cellular Biology

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The degradation of the soybean SRS4 mRNA, which encodes the small subunit of ribulose-1,5-bisphosphate carboxylase, yields a set of proximal (5 intact) and distal (3 intact) products both in vivo and in vitro. These products are generated by endonucleolytic cleavages that occur essentially in a random order, although some products are produced more rapidly than others. Comparison of sizes of products on Northern (RNA) blots showed that the combined sizes of pairs of proximal and distal products form contiguous full-length SRS4 mRNAs. When the 3 ends of the proximal products and the 5 ends of the distal products were mapped by S1 nuclease and primer extension assays, respectively, both sets of ends mapped to the same sequences within the SRS4 mRNA. A small in vitro-synthesized RNA fragment containing one cleavage site inhibited cleavage of all major sites, equivalently consistent with one enzymatic activity generating the endonucleolytic cleavage products. These products were rich in GU nucleotides, but no obvious consensus sequence was found among several cleavage sites. Preliminary evidence suggested that secondary structure could play a role in site selection. The structures of the 5 ends of the proximal products and the 3 ends of the distal products were examined. Proximal products were found with approximately equal frequency in both m 7 G cap(؉) and m 7 G cap(؊) fractions, suggesting that the endonucleolytic cleavage events occurred independently of the removal of the 5 cap structure. Distal products were distributed among fractions with poly(A) tails ranging from undetectable to greater than 100 nucleotides in length, suggesting that the endonucleolytic cleavage events occurred independently of poly(A) tail shortening. Together, these data support a stochastic endonuclease model in which an endonucleolytic cleavage event is the initial step in SRS4 mRNA degradation.RNA stability controls the amount of translatable mRNA in the cytoplasm and thus affects the level of gene expression. Changes in transcription rates more rapidly affect the steadystate levels of unstable messages than of stable ones. Therefore, an mRNA's half-life plays a significant role in regulating gene expression. Only a few general mechanisms have emerged from recent studies on the decay of mRNAs. One pathway proposed is a biphasic mechanism in which mRNAs are degraded first by removal of the 3Ј polyadenylate tail followed by degradation of the body of the message. Poly(A) tail removal is the first step in the degradation of the yeast PGK1 and MFA2 mRNAs (9). Removal of the 5Ј7-methylguanosine cap (m 7 G cap) is the second step in the degradation of the MFA2 mRNA, followed by decay of the body of the message by the 5Ј-to-3Ј

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A cell-free extract from yeast cells for studying mRNA turnover

Cited by 19 publications

References 41 publications

The rate-limiting step in yeast PGK1 mRNA degradation is an endonucleolytic cleavage in the 3'-terminal part of the coding region.

The rate-limiting step in yeast PGK1 mRNA degradation is an endonucleolytic cleavage in the 3'-terminal part of the coding region.

Yeast cells lacking 5'-->3' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5' cap structure.

Degradation of the Soybean Ribulose-1,5-Bisphosphate Carboxylase Small-Subunit mRNA, SRS4, Initiates with Endonucleolytic Cleavage

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