The poly(A)-poly(A)-binding protein complex is a major determinant of mRNA stability in vitro.

Bernstein, Philip; Peltz, Stuart W.; Ross, Jeffrey

doi:10.1128/mcb.9.2.659

Cited by 360 publications

(204 citation statements)

References 75 publications

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“…Deadenylation rates vary among mRNAs, and are often controlled by sequences in the 3Ј UTR (Muhlrad and Parker 1992;Herrick and Ross 1994;. Such 3Ј UTR elements have been suggested to provide alternative binding sites for Pab1p, shuttling it off the poly(A) tail, thereby allowing deadenylation and decay (Bernstein et al 1989). Our data argue against such models, because Pab1p binding to the 3Ј UTR stabilizes, rather than destabilizes, the mRNA.…”

Section: Discussioncontrasting

confidence: 45%

mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation

1998

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Translation and mRNA stability are enhanced by the presence of a poly(A) tail. In vivo, the tail interacts with a conserved polypeptide, poly(A) binding protein (Pab1p). To examine Pab1p function in vivo, we have tethered Pab1p to the 3 UTR of reporter mRNAs by fusing it to MS2 coat protein and placing MS2 binding sites in the 3 UTR of the reporter. This strategy allows us to uncouple Pab1p function from its RNA binding activity. We show that mRNAs that lack a poly(A) tail in vivo are stabilized by Pab1p, and that the portions of Pab1p required for stabilization are genetically distinct from those required for poly(A) binding. In addition, stabilization by Pab1p requires ongoing translation of the mRNA. We conclude that the primary, or sole, function of poly(A) with respect to mRNA stability is simply to bring Pab1p to the mRNA, and that mRNA stabilization is an intrinsic property of Pab1p. The approach we describe may be useful in identifying and assaying 3 UTR regulatory proteins, as it uncouples analysis of function from RNA binding.

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Section: Discussioncontrasting

confidence: 45%

mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation

1998

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“…Previous in vitro observations by Ross and colleagues have shown that PABP plays an important role in the stabilizing function of the poly(A) tail (6). In order to access the role of poly(A) binding proteins in our system, we performed competition experiments using poly(A) and other homopolymer RNAs.…”

Section: Resultsmentioning

confidence: 99%

“…7) may have important implications for mechanisms of regulated mRNA stabilization. This especially applies to models which suggest that destabilizing elements such as the AU-rich element can function by displacing proteins from the poly(A) tail, thereby increasing its deadenylation rate (6). The construct containing an internal poly(A) tract may be comparable to the situation in which the terminal poly(A) binding protein is removed from a full-length poly(A) tail.…”

Section: Discussionmentioning

confidence: 99%

The Poly(A) Tail Inhibits the Assembly of a 3′-to-5′ Exonuclease in an In Vitro RNA Stability System

Ford

Bagga

Wilusz

1997

Molecular and Cellular Biology

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We have developed an in vitro system which faithfully reproduces several aspects of general mRNA stability. Poly(A) ؊ RNAs were rapidly and efficiently degraded in this system with no detectable intermediates by a highly processive 3 -to-5 exonuclease activity. The addition of a poly(A) tail of at least 30 bases, or a 3 histone stem-loop element, specifically stabilized these transcripts. Stabilization by poly(A) required the interaction of proteins with the poly(A) tail but did not apparently require a 3 OH or interaction with the 5 cap structure. Finally, movement of the poly(A) tract internal to the 3 end caused a loss of its ability to stabilize transcripts incubated in the system but did not affect its ability to interact with poly(A) binding proteins. The requirement for the poly(A) tail to be proximal to the 3 end indicates that it mediates RNA stability by blocking the assembly, but not the action, of an exonuclease involved in RNA degradation in vitro.The relative stability of RNA transcripts plays a key role in determining their steady-state levels as well as the rate of mRNA induction following a transcriptional stimulus (30). Mutations which affect transcript stability can have a very significant impact on regulated gene expression (26,33). The mechanism of regulated turnover of mammalian mRNA, however, is largely unknown. Terminal structures, namely, the 5Ј cap and 3Ј poly(A) tail, serve as general stabilizing elements found on most mRNAs (7,14). Several internal sequences, such as an AU-rich element (10) or nonsense codons (5), which functionally shorten the half-lives (t 1/2 ) of mRNAs have been identified. Furthermore, an internal element which stabilizes ␣ globin mRNA has recently been identified (38). Elucidating how the general and regulatory elements interact to determine the functional t 1/2 of mRNAs is vital to understanding the mechanism of RNA stability as a regulator of gene expression.The poly(A) tail, a posttranscriptional modification of the 3Ј end of all nonhistone mRNAs, is initially formed as a 150-to 200-base homopolymer (19) which assembles multiple molecules of a poly(A) binding protein (PABP) (13). The tail is a dynamic structure which serves as a mediator through which several important cellular processes including the initiation of translation (31), nucleocytoplasmic transport (18), and mRNA stability (7), are regulated. Most mRNAs are rapidly degraded following deadenylation of their 3Ј ends to approximately 30 adenylate residues (8,21,27,34). Many destabilizing elements appear to act by increasing the rate of deadenylation (11). The disruption of poly(A) tail function, therefore, appears to be the rate-limiting step in mRNA turnover. Following deadenylation, mRNA degradation can occur through a variety of exoand/or endonucleolytic pathways (4). The difficulty in identifying intermediates in mammalian mRNA turnover has significantly hindered attempts to probe further into mechanistic aspects of the turnover process.Mechanistic questions in mammalian systems are usually best ...

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“…Polyadenylation has very different effects on RNA stability in different systems. For example, in eukaryotic cytoplasm, the poly(A) tail in conjunction with poly(A)-binding proteins increases the stability of many mRNAs (2)(3)(4)(5). On the contrary, in bacteria and chloroplasts, poly(A) tails act as mRNA destabilizing elements (6)(7)(8).…”

Section: Introductionmentioning

confidence: 99%

Targeted depletion of a mitochondrial nucleotidyltransferase suggests the presence of multiple enzymes that polymerize mRNA 3′ tails in Trypanosoma brucei mitochondria

Kao

Read

2007

Molecular and Biochemical Parasitology

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Polyadenylation plays an important role in regulating RNA stability in Trypanosoma brucei mitochondria. To date, little is known about the enzymes responsible for the addition of mRNA 3′ tails in this system. In this study, we characterize a trypanosome homolog of the human mitochondrial poly(A) polymerase, which we term kPAP2. kPAP2 is mitochondrially localized and expressed in both bloodstream and procyclic form trypanosomes. Targeted gene depletion using RNAi showed that kPAP2 is not required for T. brucei growth in either bloodstream or procyclic life stages, nor is it essential for differentiation from bloodstream to procyclic form. We also demonstrate that steady state abundance of several mitochondrial RNAs was largely unaffected upon kPAP2 downregulation. Interestingly, mRNA 3′ tail analysis of several mRNAs from both life cycle stages in uninduced kPAP2 RNAi cells demonstrated that tail length and uridine content are both regulated in a transcript-specific manner. mRNA-specific tail lengths were maintained upon kPAP2 depletion. However, the percentage of uridine residues in 3′ tails was increased, and conversely the percentage of adenosine residues was decreased, in a distinct subset of mRNAs when kPAP2 levels were downregulated. Thus, kPAP2 apparently contributes to the incorporation of adenosine residues in 3′ tails of some, but not all, mitochondrial mRNAs. Together, these data suggest that multiple nucleotidyltransferases act on mitochondrial mRNA 3′ ends, and that these enzymes are somewhat redundant and subject to complex regulation.

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The poly(A)-poly(A)-binding protein complex is a major determinant of mRNA stability in vitro.

Abstract: Using an in vitro mRNA decay system, we investigated how poly(A) and its associated poly(A)-binding protein (PABP)

Cited by 360 publications

References 75 publications

mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation

mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation

The Poly(A) Tail Inhibits the Assembly of a 3′-to-5′ Exonuclease in an In Vitro RNA Stability System

Targeted depletion of a mitochondrial nucleotidyltransferase suggests the presence of multiple enzymes that polymerize mRNA 3′ tails in Trypanosoma brucei mitochondria

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