Article

Messenger RNA decay in mammalian cells: the exonuclease perspective.

Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, NJ 07101, USA.
Cell biochemistry and biophysics (impact factor: 3.34). 02/2004; 41(2):265-78. DOI:10.1385/CBB:41:2:265 pp.265-78
Source: PubMed

ABSTRACT The majority of messenger RNA (mRNA) decay in mammalian cells appears to be the work of a series of RNA exoribonucleases. A set of multiple poly(A)-specific deadenylases has been identified, some, if not most, of which are likely to play a role in the key first step of mRNA turnover--the regulated shortening of the poly(A) tail. After deadenylation, the transcript likely gets degraded by either a 5'-to-3' or a 3'-to-5' exonucleolytic pathway. Interestingly, multiple exonucleases have been identified for both of these pathways that appear to form multicomponent complexes with diverse roles in cellular biology. Therefore these enzymes appear not only to be important components of the mRNA turnover machinery, but also may function in a networked fashion in the post-transcriptional control of gene expression.

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    Dataset: MCB 2007
    Francesca Voza, Nader Ezzeddine, Hanh T. Nguyen, and Manfred Frasch
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    Dataset: Human TOB, an Antiproliferative Transcription Factor, Is a Poly(A)-Binding Protein-Dependent Positive Regulator of Cytoplasmic mRNA Deadenylation
    Nader Ezzeddine, Tsung-Cheng Chang, Wenmiao Zhu, Akio Yamashita, Chyi-Ying A Chen, Zhenping Zhong, Yukiko Yamashita, Dinghai Zheng, Ann-Bin Shyu
    [show abstract] [hide abstract]
    ABSTRACT: In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases medi-ated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenyl-ation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these find-ings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenyl-ation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3 poly(A) tail-PABP complex. Deadenylation is the first major step that triggers mRNA decay in eukaryotic cells (reviewed in references 19, 41, and 44). Computational modeling of eukaryotic mRNA turnover indicates that changes in levels of mRNA are highly leveraged to the rate of deadenylation (8). The importance of deadenyl-ation in regulating mammalian mRNA turnover can be ob-served in several modes of mRNA decay, including decay di-rected by AU-rich elements in the 3 untranslated region (4, 10), the rapid decay mediated by destabilizing elements in protein-coding regions (9, 23), the surveillance mechanism that detects and degrades nonsense-containing mRNA (11), and the decay directed by microRNA (59). Shortening of the 3 poly(A) tail also plays a critical role in rendering mRNAs nontranslatable (26, 46, 58), thus inactivating gene expression. In spite of the importance of deadenylation, relatively little is known about the mechanisms that control it. Recent progress in identifying key mammalian poly(A) nucleases involved in deadenylation (1, 6, 13, 16, 20, 38, 53, 55) has offered the opportunity to examine the regulation of de-adenylation and to characterize the participating regulatory proteins. In mammalian cells, shortening of the poly(A) tail is mediated by the consecutive activities of two different poly(A) nuclease complexes (61). During the first phase, PAN2, pre-sumably complexed with PAN3 (53, 61), shortens the poly(A) tails to 110 A nucleotides. In the second phase, CCR4, pre-sumably complexed with CAF1 (6, 55, 61), further shortens the poly(A) tail to oligo(A). Decapping mediated by the DCP1-DCP2 complex (36, 54, 56) was found to occur after either the first or the second phase of deadenylation (61). To identify the potential regulatory factors involved in mam-malian deadenylation, we have carried out literature and da-tabase searches with a focus on proteins that have the potential to interact with a poly(A) nuclease and/or the cytoplasmic poly(A)-binding protein (PABPC1). A family of antiprolifera-tive genes, termed the tob/btg family (reviewed in references 24 and 39), emerged from the searches, because they contain a highly conserved N-terminal domain that can interact with CAF1 (7, 42). In humans, this family consists of six members: tob, tob2, ana, pc3b, btg1, and btg2, among which tob and tob2 also encode a C-terminal domain with two putative PABP-interacting motifs (24, 39). Increasing evidence suggests that TOB proteins are involved in negative control of cell growth and can function as tumor suppressors (25, 49, 62). Moreover, TOB is highly expressed in anergic T-cell clones and in un-stimulated peripheral blood T lymphocytes (52). The ability of TOB to maintain T-cell quiescence is thought to be due to its modulation of transcription (52). Despite the fact that TOB proteins have been known for a decade to function in antipro-liferation and potentially in transcriptional control, the bio-chemical and molecular mechanisms by which they exert their functions remains unclear. In this study, our results revealed that TOB proteins
  • Source
    Article: Human TOB, an Antiproliferative Transcription Factor, Is a Poly(A)-Binding Protein-Dependent Positive Regulator of Cytoplasmic mRNA Deadenylation
    Nader Ezzeddine, Tsung-Cheng Chang, Wenmiao Zhu, Akio Yamashita, Chyi-Ying A Chen, Zhenping Zhong, Yukiko Yamashita, Dinghai Zheng, Ann-Bin Shyu
    [show abstract] [hide abstract]
    ABSTRACT: In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases medi-ated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenyl-ation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these find-ings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenyl-ation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3 poly(A) tail-PABP complex. Deadenylation is the first major step that triggers mRNA decay in eukaryotic cells (reviewed in references 19, 41, and 44). Computational modeling of eukaryotic mRNA turnover indicates that changes in levels of mRNA are highly leveraged to the rate of deadenylation (8). The importance of deadenyl-ation in regulating mammalian mRNA turnover can be ob-served in several modes of mRNA decay, including decay di-rected by AU-rich elements in the 3 untranslated region (4, 10), the rapid decay mediated by destabilizing elements in protein-coding regions (9, 23), the surveillance mechanism that detects and degrades nonsense-containing mRNA (11), and the decay directed by microRNA (59). Shortening of the 3 poly(A) tail also plays a critical role in rendering mRNAs nontranslatable (26, 46, 58), thus inactivating gene expression. In spite of the importance of deadenylation, relatively little is known about the mechanisms that control it. Recent progress in identifying key mammalian poly(A) nucleases involved in deadenylation (1, 6, 13, 16, 20, 38, 53, 55) has offered the opportunity to examine the regulation of de-adenylation and to characterize the participating regulatory proteins. In mammalian cells, shortening of the poly(A) tail is mediated by the consecutive activities of two different poly(A) nuclease complexes (61). During the first phase, PAN2, pre-sumably complexed with PAN3 (53, 61), shortens the poly(A) tails to 110 A nucleotides. In the second phase, CCR4, pre-sumably complexed with CAF1 (6, 55, 61), further shortens the poly(A) tail to oligo(A). Decapping mediated by the DCP1-DCP2 complex (36, 54, 56) was found to occur after either the first or the second phase of deadenylation (61). To identify the potential regulatory factors involved in mam-malian deadenylation, we have carried out literature and da-tabase searches with a focus on proteins that have the potential to interact with a poly(A) nuclease and/or the cytoplasmic poly(A)-binding protein (PABPC1). A family of antiprolifera-tive genes, termed the tob/btg family (reviewed in references 24 and 39), emerged from the searches, because they contain a highly conserved N-terminal domain that can interact with CAF1 (7, 42). In humans, this family consists of six members: tob, tob2, ana, pc3b, btg1, and btg2, among which tob and tob2 also encode a C-terminal domain with two putative PABP-interacting motifs (24, 39). Increasing evidence suggests that TOB proteins are involved in negative control of cell growth and can function as tumor suppressors (25, 49, 62). Moreover, TOB is highly expressed in anergic T-cell clones and in un-stimulated peripheral blood T lymphocytes (52). The ability of TOB to maintain T-cell quiescence is thought to be due to its modulation of transcription (52). Despite the fact that TOB proteins have been known for a decade to function in antipro-liferation and potentially in transcriptional control, the bio-chemical and molecular mechanisms by which they exert their functions remains unclear. In this study, our results revealed that TOB proteins
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Keywords

3'-to-5' exonucleolytic pathway
 
diverse roles
 
form multicomponent complexes
 
key first step
 
messenger RNA
 
mRNA turnover machinery
 
multiple exonucleases
 
multiple poly(A)-specific deadenylases
 
networked fashion
 
pathways
 
post-transcriptional control
 
transcript likely