Interactions of CCCH Zinc Finger Proteins with mRNA

Lai, Wi S.; Carballo, E.; Thorn, Judith M.; Kennington, Elizabeth A.; Blackshear, Perry J.

doi:10.1074/jbc.m001696200

Cited by 329 publications

(170 citation statements)

References 31 publications

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“…As with classical loss-of-function tumor suppressor genes (Hanahan and Weinberg, 2000;Knudson, 2000), TTP or a closely related gene may be mutated in the tumor cells and transfection restores the missing function. We have sequenced the entire coding region of TTP and BRF1, a structurally related AREbinding zinc-finger protein that also regulates cytokine mRNA turnover (Lai et al, 2000;Stoecklin et al, 2002). In V2D1 tumor cells, no mutation was detected in either gene, and transcripts were of expected size and expressed at the same levels as in parental PB-3c cells (data not shown).…”

Section: Discussionmentioning

confidence: 99%

“…The mechanism of mRNA stabilization is likely to involve ARE-binding proteins as adaptors between the signalling cascade and the decay machinery (Carballo et al, 2001;Mahtani et al, 2001). ARE-binding proteins with assigned functions in vivo include TTP, butyrate response factor-1 (BRF1) and AUF1, all of which promote degradation, as well as HuR that exerts an mRNA stabilizing effect (Carballo et al, 1998;Fan and Steitz, 1998;Peng et al, 1998;Loflin et al, 1999;Lai et al, 2000;Stoecklin et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

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A novel mechanism of tumor suppression by destabilizing AU-rich growth factor mRNA

et al. 2003

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The occurrence of pathologically stable mRNAs of protooncogenes, growth factors and cyclins has been proposed to contribute to experimental and human oncogenesis. In normal resting cells, mRNAs containing an AU-rich element (ARE) in their 3 0 untranslated region are subjected to rapid degradation. Tristetraprolin (TTP) is an RNA-binding zinc-finger protein that promotes decay of ARE-containing mRNAs. Here we report that TTP acts as a potent tumor suppressor in a v-H-ras-dependent mast cell tumor model, where tumors express abnormally stable interleukin-3 (IL-3) mRNA as part of an oncogenic autocrine loop. Premalignant v-H-ras cells were transfected with TTP and injected into syngeneic mice. TTP expression delayed tumor progression by 4 weeks, and late appearing tumors escaped suppression by loss of TTP. When transfected into a fully established tumor line, TTP reduced cloning efficiency in vitro and growth of the inoculated cells in vivo. Transgenic TTP interfered with the autocrine loop by enhancing the degradation of IL-3 mRNA with concomitant reduction of IL-3 secretion. Our data establish the ARE as an antioncogenic target in a model situation, underline the importance of mRNA stabilization in oncogenesis and show for the first time that tumor suppression can be achieved by interfering with mRNA turnover.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A novel mechanism of tumor suppression by destabilizing AU-rich growth factor mRNA

et al. 2003

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“…In vitro selection of randomized RNA with recombinant TTP had yielded RNAs very similar to the prototype mRNA instability element, UUAUUUA(U/A)(U/A) (57). Comparison of the full-length TTP protein to its isolated RNA-binding domain (a 77-amino acid region containing the tandem TTP zinc fingers) has indicated that the RNA-binding ability and indeed the ability to destabilize tumor necrosis factor ␣ mRNA are maintained in the isolated RNA-binding domain (58). poly(U) can be used to deplete TTP from cellular extracts, thereby removing its mRNA destabilizing activity, suggesting this protein can bind well to poly(U) (17); however, competition experiments have suggested that poly(U) is not a strong target for TTP (59).…”

Section: Figmentioning

confidence: 99%

Characterization of the Interaction between Neuronal RNA-binding Protein HuD and AU-rich RNA

Park-Lee

Kim

Laird-Offringa

2003

Journal of Biological Chemistry

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Hu proteins have been shown to bind to AU-rich elements (AREs) in the 3 -untranslated region of unstable mRNAs. They can thereby inhibit the decay of labile transcripts by antagonizing destabilizing proteins that target these AU-rich sequences. Here we examine the sequence preferences of HuD to elucidate its possible role in counteracting mRNA decay. Using repeats of the prototype destabilizing sequence UU(AUUU) n AUU, we show that all three HuD RNA-binding domains participate in binding to AU-tracts that can be as short as 13 residues, depending on the position of the remaining As. Removal of the A residues, resulting in a poly(U)-tract, increased the affinity of HuD for RNA, suggesting that the presence of As in destabilizing elements might favor the recruitment of other proteins and/or prevent HuD from binding too tightly to AREs. In vitro selection experiments with randomized RNAs confirmed the preference of HuD for poly(U). RNA binding analysis of the related protein HuB showed a similar preference for poly(U). In contrast, tristetraprolin, an mRNA destabilizing protein, strongly prefers AU-rich RNA. Many labile mRNAs contain U-tracts in or near their AREs. Individual AREs may thus differentially affect mRNA halflife by recruiting a unique complement of stabilizing and destabilizing factors.

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“…Human embryonic kidney 293 cells (referred to as 293 cells) were maintained, and transient transfection of 1.2 ϫ 10 6 cells with plasmid constructs in calcium phosphate precipitates was performed as described (20). In some experiments, pXGH5 (Nichols Institute Diagnostics, San Juan Capistrano, CA) was cotransfected to monitor transfection efficiency.…”

Section: Transfection Of Human Embryonic Kidney 293 Cells and Northermentioning

confidence: 99%

“…Apparently normal binding still occurs when the TTP protein is truncated to a 77-amino acid fragment containing the TZF domain and seven flanking residues on either side (20). By investigating the binding of RNA to TZF domains from various CCCH proteins, we have begun to characterize the extent of natural variation that can be tolerated and still maintain RNA-binding activity (20). In the present study, we have extended these findings by generating and testing a series of site-directed mutants of the TTP TZF domain.…”

mentioning

confidence: 99%

Interactions of CCCH Zinc Finger Proteins with mRNA

Lai¹,

Kennington²,

Blackshear³

2002

Journal of Biological Chemistry

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133

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Tristetraprolin (TTP), the prototype of a small family of CCCH tandem zinc finger (TZF) domain proteins, is a physiological stimulator of instability of the mRNAs encoding tumor necrosis factor-␣ and granulocyte/macrophage colony-stimulating factor in certain cell types. TTP stimulates mRNA turnover after binding to class II AU-rich elements (AREs) within the 3-untranslated regions of both mRNAs. In turn, this binding is dependent upon the key CCCH residues in the TZF domain. To evaluate other primary sequence requirements for ARE binding in this novel mRNA-binding domain, we mutated many of the conserved residues within the TZF domain of human TTP and evaluated the effects of these mutations on RNA binding in a cell-free system and TTP-induced mRNA instability in cell transfection experiments. These mutations revealed a number of conserved amino acids that were required for binding and begin to define the primary protein sequence requirements for this novel mRNA-binding motif. Unexpectedly, all of the point mutations that prevented TTP binding to RNA also caused an increase in steady-state levels of ARE-containing mRNAs in cell transfection experiments. Actinomycin D experiments suggested that this effect was due to inhibition of mRNA turnover. Although expression of the mutant form of TTP could also inhibit the destruction of tumor necrosis factor-␣ mRNA by wild-type TTP, the primary mechanism did not involve heterodimerization with wild-type TTP because the 293 cells used in these studies express no detectable endogenous TTP. These data suggest that TTP may act, at least in part, by physically interacting with an enzyme activity or protein complex and functionally stimulating its ability to deadenylate class II ARE-containing mRNAs.The CCCH zinc finger motif has been found in proteins from organisms ranging from man to yeast (1-11). A small subset of these proteins contain two CCCH zinc fingers with the following characteristics. The fingers are 18 amino acids apart; they have the internal spacing CX 8 CX 5 CX 3 H, where X indicates variable amino acids; and both fingers have a highly conserved lead-in sequence, R(K)YKTEL. Three such proteins have been identified to date in mammals, with a fourth member recently discovered in Xenopus and fish (2, 12, 13). We will refer to this small group of proteins as the CCCH tandem zinc finger (TZF) 1 domain proteins.Our group has begun to elucidate a function for this group of proteins by analyzing the phenotype of mice deficient in tristetraprolin (TTP), the prototype of this group of proteins. These animals develop a complex phenotype consisting of cachexia, dermatitis, conjunctivitis, destructive arthritis, myeloid hyperplasia, and autoimmunity (14). Virtually all aspects of this phenotype can be prevented by the repeated injection of antibodies to tumor necrosis factor-␣ (TNF␣), implicating an effective excess of circulating TNF␣ in the pathogenesis of this condition (14). This was confirmed by studies showing that the phenotype can be largely prevented by interbreed...

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Interactions of CCCH Zinc Finger Proteins with mRNA

Cited by 329 publications

References 31 publications

A novel mechanism of tumor suppression by destabilizing AU-rich growth factor mRNA

A novel mechanism of tumor suppression by destabilizing AU-rich growth factor mRNA

Characterization of the Interaction between Neuronal RNA-binding Protein HuD and AU-rich RNA

Interactions of CCCH Zinc Finger Proteins with mRNA

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