Multiple functions of tristetraprolin/TIS11 RNA-binding proteins in the regulation of mRNA biogenesis and degradation

Abstract

Members of the tristetraprolin (TTP/TIS11) family are important RNA-binding proteins initially characterized as mediators of mRNA degradation. They act via their interaction with AU-rich elements present in the 3′UTR of regulated transcripts. However, it is progressively appearing that the different steps of mRNA processing and fate including transcription, splicing, polyadenylation, translation, and degradation are coordinately regulated by multifunctional integrator proteins that possess a larger panel of functions than originally anticipated. Tristetraprolin and related proteins are very good examples of such integrators. This review gathers the present knowledge on the functions of this family of RNA-binding proteins, including their role in AU-rich element-mediated mRNA decay and focuses on recent advances that support the concept of their broader involvement in distinct steps of mRNA biogenesis and degradation.

Overview of the tristetraprolin (TTP/TIS11) family

Description of tristetraprolin (TTP) and related proteins started in the early 1990s with the cloning of an immediate–early response gene induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in murine fibroblasts, which was thereafter named TPA-induced sequence 11 (TIS11) [1]. At that time, several other groups that were deciphering the complex transcriptional response of various cell types to stimulation by mitogens reported the induction of mRNAs sharing structural similarities with TIS11 and contributed to the definition of a new family of early response genes, the TTP/TIS11 family [2–6].

In mammals, beside TTP/TIS11 (HGNC alias: ZFP36), which is the most thoroughly studied member, the family contains two other members called TIS11b/BRF1 (HGNC alias: ZFP36L1) and TIS11d/BRF2 (HGNC alias: ZFP36L2). In addition to these well-described members, rodents possess an additional member (HGNC alias: ZFP36L3) specifically expressed in the placenta [7, 8]. Like TTP, all members of the family are characterized by the presence in their coding sequence of a very particular tandem zinc-finger domain (TZF), which can be found in almost all branches of eukaryotic evolution from lower unicellular eukaryotes to invertebrates and mammals [9–12]. This structural domain is composed of a double zinc-finger motif of the CCCH type, each one preceded by a leader sequence, and bears the RNA-binding property (see Fig. 1a).

Fig. 1.

Similarities in the protein domains of the TTP family members. a Sequence of the tandem zinc-finger domain (TZF), which is characteristic of the TTP family. TIS11b amino-acid residues inside the TZF share 70 and 90 % of sequence identity with TIS11d and TTP/TIS11, respectively. b Remarkable features in TTP/TIS11, TIS11b/BRF1, and TIS11d/BRF2 protein. Tristetraprolin has been named after the particular repeated motif (PPPPG) of the protein. Each protein of the family contains a nuclear localization signal (NLS) inside the much-conserved TZF domain and a conserved post-TZF domain of unknown function. Nuclear export signals (NES) are located in the C-termini of TIS11b and TIS11d and in the N-terminus of TTP. The N-terminal domains of TIS11b and TIS11d are closely related, but differ from that of TTP

It is worth mentioning that the TZF domain is of very high sequence homology between the TTP family members, whereas the N-terminal and C-terminal regions differ much more. Figure 1b, which depicts the similarities and differences in the sequences of the human members of the TTP family illustrates the remarkable features of these proteins. Indeed, in addition to the TZF motif, only two other domains of relative high homology are present in the three proteins. One is a stretch of 14 amino acids that contains a functional nuclear export sequence (NES). This sequence is C-terminal in the TIS11b and TIS11d sequences, whereas for TTP/TIS11, the NES is located instead in the N-terminus of the protein. The second similarity motif is located immediately downstream of the TZF domain and is composed of the PxLxxSxSFxGxPS sequence, where x represents one member of a closely related family of amino acids. The exact function of this domain is unknown, but contains (at least for TTP and TIS11b) a binding site for 14-3-3 proteins [13, 14]. Moreover, the presence of three serine and one proline residues at both ends suggests a loop conformation for this motif. In fact, sequence alignments suggest that TIS11b and TIS11d are more closely related to each other than to TTP. This is particularly true at the N-terminal part of these proteins, where domain similarities between TIS11b and TIS11d define a specific submotif in the protein family (PFAM PF04553). Besides these similarities, the weaker protein homology observed outside the TZF motif suggests that the specific protein–protein interactions mediating the distinct functions and regulations of the family members are borne by these divergent domains.

The notion that these proteins are not physiologically redundant is clearly illustrated by the differences in the phenotypes of the knock-out mice generated by ubiquitous genetic deletion of the distinct family members (presented in Table 1). TTP/TIS11 KO mice appear normal at birth, but rapidly develop a complex syndrome of cachexia, arthritis, and general inflammation, which can be almost completely prevented by injection of antibodies against TNF-α [15]. In contrast, disruption of the TIS11b/BRF1 gene induces embryonic lethality around E10.5 with neural tube abnormalities, failure of chorioallantoic fusion, and angiogenesis defects [16, 17]. Concerning TIS11d, deletion of the 29 N-terminal amino acids of the protein results in female infertility, whereas the complete inactivation of the gene induces lethality within 2 weeks after birth due to diverse hemorrhages, probably caused by defective definitive hematopoiesis [18–20]. These studies showed that (at least for some of their physiological functions) members of the TIS11 family cannot compensate for each other. However, compensation between TIS11b and TIS11d proteins has been particularly well described in a recent study, where inducible and concomitant deletions of both TIS11b and TIS11d genes was realized in the thymus during thymopoiesis. This was reported to trigger lymphoblastic leukemia, whereas disruption of either one of these genes was not sufficient to induce this phenotype [21]. Altogether, these studies suggest that possible compensation might exist when two or three members are expressed in the same organ during the same developmental window. The activities of the TTP family proteins must therefore be analyzed in a cell- and context-dependent manner.

Table 1.

Phenotypes of the knock-out mice for the three members of the TTP family

8Name (alias)	Reference	Lethality	Main characteristics	Comments
ZFP36 (TTP,TIS11,G0S24, NUP475, RNF162A)	[15]	Post-natal (depending on the severity of the syndrome)	Inflammatory syndrome (cachexia, arthritis)	Rescued by anti-TNF-α antibodies injection first identification of TTP implication in AU-rich mediated decay
ZFP36L1 (BRF1, TIS11b, Berg36, ERF-1, ERF1, cMG1)	[17]	E11	Neural tube and chorioallantoic fusion defects
	[16]	E10.5	Neural tube and vascular angiogenesis defects	Identification of potential role of Tis11b/BRF1 in the control of AU-rich-mediated translation
ZFP36L2 (TIS11d, BRF2, ERF-2, ERF2, RNF162C)	[18, 19]	None	Female infertility	Knock-out is incomplete resulting in an amino-terminal truncation of the protein
	[20]	2 weeks after birth	Definitive hematopoiesis deficiency	Identification of 293 up-regulated transcripts in KO fetal livers, but no apparent effect on mRNA stability in KO fibroblasts
ZFP36L1-ZFP36L2-inducible double KO in the thymus	[21]	90 % of lethality by 6 months of age	Lymphoblastic, leukemia	First direct identification of malignant transformation due to deficiency in Tis11 proteins expression

Localization, expression, and regulation of TTP and related proteins

TTP and related proteins are nucleocytoplasmic shuttling proteins. They all contain a specific nuclear localization sequence (NLS) located between the two zinc fingers [22, 23]. In addition to these import sequences, nuclear export signals are present in the N-terminus of TTP or in the C-termini of both TIS11b and TIS11d. Nucleocytoplasmic shuttling of TTP family members is dependent on these signals and on the activity of the nuclear export receptor CRM1 [23]. Moreover, TTP directly associates with the nucleoporin NUP214 [24]. Interestingly, a modification of the C-terminal part of ZFP36L3, the murine-specific member of the family specifically expressed in the placenta, appears to result in the loss of its shuttling capacity and in an enforced cytoplasmic localization [8]. Subcellular localization studies indicated that the nuclear or cytoplasmic localizations of TTP-related proteins differ from one cell type to another and are regulated by extracellular signals, which thereby modulate their respective functions [25–29].

Post-translational modifications strongly contribute to the regulation of TTP family protein activities. TTP family members are phosphorylated on several distinct sites in response to multiple signaling pathways [28, 30–34]. Initially, it has been speculated that phosphorylation of TTP-related proteins would modulate their RNA-binding properties and therefore their mRNA-destabilizing properties. Nevertheless, experimental results have been contradictory, with some studies showing an increase in TTP RNA-binding capacities in phosphatase-treated cellular extracts, whereas in vitro phosphorylation of either TTP or TIS11b had no effect on RNA binding [35–37]. To date, phosphorylations of either TTP or TIS11b have been reported to impair their mRNA decay-promoting activities (reviewed in [38]). The most extensively studied kinases are the p38-MAPK (p38-mitogen-activated protein kinase) and its downstream target MAPK-activated protein kinase 2 (MK2), both of which appear to play a pivotal role in the regulation of ARE-mediated mRNA decay (AMD). Inhibition of p38 MAPK signaling pathway has been reported to promote AMD [25], whereas, on the contrary, expression of constitutively active upstream activators of p38 MAPK increases stability of ARE-containing transcripts [39]. p38 and MK2 phosphorylate TTP protein at two critical sites, Ser⁵² and Ser¹⁷⁸, leading to an increased TTP protein stability and TTP cytoplasmic localization [13, 25, 32]. Similarly, phosphorylations of TIS11b at Ser⁹² and Ser²⁰³ by protein kinase B (PKB) and MK2 stabilize TIS11b protein [14, 37, 40]. For both TTP and TIS11b, the above-mentioned phosphorylated serines are docking sites for 14-3-3 protein isoforms [14, 41]. 14-3-3 protein binding can indeed modulate the cytoplasmic localization and protein stability of both TTP and TIS11b [25, 40]. Moreover, interaction with 14-3-3 inhibits TTP protein dephosphorylation by protein phosphatase 2A (PP2A) and therefore modulates TTP function [42]. It has also been shown that phosphorylation of TTP favors its ubiquitinylation and that TTP and TIS11b proteins are regulated by proteasome activity depending on their phosphorylation status [25, 40, 43, 44]. Phosphorylations of TTP protein also have a direct impact on its mRNA-destabilizing activity by preventing the recruitment of mRNA deadenylases onto the target mRNA. This inhibitory effect appears to be, at least partially, independent of 14-3-3 protein sequestration and unrelated to TTP RNA-binding [45, 46]. Overall, the observation that TTP or TIS11b protein stability was regulated by the same phosphosites which were originally identified as regulatory sites for their mRNA decay-promoting activity revealed an unexpected link between TTP or TIS11b protein turnover and their function in AMD.

The signaling pathways that regulate expression, localization, degradation, or function of TTP family members are well described in recent reviews [38, 47–49] and are illustrated in Fig. 2. It has been speculated that, by regulating the stability, localization and function of TTP family proteins, distinct signaling pathways can coordinate the decay rate of specific subsets of target mRNAs and therefore the expression of the corresponding proteins [32, 40].

Fig. 2.

Signaling pathways acting on TTP and TIS11b/BRF1 proteins. Schematic representation of the diverse signaling pathways modulating either TTP/TIS11 or TIS11b/BRF1 protein expression, localization, degradation, or capacity to recruit mRNA decay factors

Not only AU-rich RNA binding proteins

AU-rich elements (AREs) are consensus mRNA cis-acting sequences containing a high proportion of adenylate or uridylate bases, which have been previously described as RNA-destabilizing motifs [50]. These regulatory motifs have been characterized in early response genes or short-lived mRNAs and classified depending on the absence (class III) or the presence of isolated (Class I) or clustered (Class II) pentameric “AUUUA” motifs [51]. Since then, computational analyses have been used to cluster human and mouse ARE-containing transcripts. A comprehensive database (ARED database, [52, 53]) has been built based on the presence in the transcript 3′-UTR of the minimal motif WWWT[ATTTA]TWWW (with one allowed mismatch outside of the bracketed region and where W is either an adenylate or an uridylate). Indeed, these analyses showed that almost 8 % of the human transcriptome contain AREs in their mRNA 3′-UTRs and that conservation between species is high for a large portion of them (ARED database, [52, 53]). This suggests that the role of AMD in gene regulation is widespread among the living species. Recently, an algorithm (termed AREScore) has been described, which uses the only core “AUUUA” motif and its surrounding sequence characteristics to identify and score the chance of AMD regulation for a given mRNA [54]. Interestingly, this approach, which uses a less-stringent criterion, can identify potential ARE-controlled mRNAs that are not integrated in the ARED database.

As mentioned above, TTP and its family members have been shown to specifically bind AREs via their highly conserved double zinc-finger domain [55]. Identification and analysis of RNA sequences selectively bound by TTP in RNA SELEX experiments confirmed this preferential recognition of the core AUUUA element and showed a strong specificity for the extended motif UAUUUAU [56]. Further studies confirmed that this extended motif is sufficient to allow formation of an RNA–protein complex [57]. Indeed, NMR structures of the TIS11d double zinc finger in complex with RNA showed that each zinc finger binds symmetrically to adjacent 5′-UAUU-3′ sub-sites on the single-stranded RNA [58]. Of note, a previous NMR structure analysis of the single first zinc finger of TTP showed slight differences in folding, probably due to the absence of the RNA ligand [59, 60].

Despite their high specificity for AREs, it has been suggested that TTP family members can bind non-ARE-like motifs [61]. When identifying TTP mRNA targets in human dendritic cells, Emmons et al. [61] characterized a subset of mRNA bound and regulated by TTP which contained a CTTGTG motif. They further demonstrated that deletion of a 36-nt portion of the 3′-UTR containing this motif was sufficient to abrogate the TTP response in reporter gene experiments. Moreover, within the same year, a genome-wide screening identified 137 potential TTP-bound mRNAs, with only some of them containing well-defined AREs, suggesting that TTP binding might not be restricted to AREs or might associate with other mRNA-binding proteins that target distinct sequences [62].

In addition to these experiments showing binding to non-ARE sequences, some reports also suggested that TTP can modulate mRNA expression independently of its mRNA-binding activity. Indeed, regulation of iNOS mRNA stability seems to involve a complex interplay between several ARE-binding proteins including HuR and KH-type splicing regulatory protein (KSRP) [63]. A direct interaction between TTP and KSRP inhibits the recruitment of the latter onto ARE and therefore prevents mRNA degradation via the exosome [64]. Moreover, it has recently been shown that binding of different isoforms of AUF1 to TTP via its zinc-finger domain can modulate TTP-binding affinity to its target mRNA sequence, thereby promoting TTP-dependent mRNA degradation [65]. These studies suggest that TTP may also act indirectly on non-ARE sequences by participating in a network of interactions with other RNA-binding proteins.

Functions of the TTP/TIS11 proteins in mRNA destabilization

mRNA destabilization historically remains the best-characterized function of the TTP family members. The first description of TTP involvement in transcript degradation came from the description of the mouse phenotype resulting from its genetic deletion [66]. In this study, Blackshear and collaborators showed that the induction of TNF-α production observed in the TTP-KO mice was due to increased TNF-α mRNA stability in macrophages. Further studies from the same group indicated that TTP and related proteins are able to bind to TNF-α AREs via their double zinc-finger motif, to promote deadenylation of the TNF-α transcript and thereby to induce mRNA destabilization [55, 67–69]. In vitro experiments showed that TTP and its family members can promote deadenylation of ARE-containing mRNAs by the deadenylase PARN, while having no impact on ARE-devoid transcripts [70]. Besides this deadenylation-promoting effect, no direct interaction could be identified between TTP and PARN, suggesting that modification of the RNA conformation induced upon TTP binding to AREs might favor the recruitment of this deadenylase [45, 70, 71].

On the other hand, more recently, TTP and TIS11b/BRF1 have been found to be involved in the recruitment of several mRNA decay enzymes (including deadenylases, decapping enzyme, and exosome complex components) onto ARE-containing transcripts [71]. Coimmunoprecipitation assays using TTP or TIS11b as bait revealed an interaction of these proteins with the CCR4 deadenylase. CCR4 is a component of a large cytoplasmic deadenylation complex containing another deadenylase, CAF1, and the scaffolding protein NOT1. Later on, the involvement of the CCR4–CAF1 complex in TTP-dependent deadenylation was further confirmed by the identification of an interaction between TTP and CAF1 by GST pull-down assays and by the observation that depletion of either the CCR4 or CAF1 protein abrogates in vitro TTP-directed deadenylation [46]. It appears that TTP is associated with multiple deadenylases as reported by Clement and collaborators [45], even though they confirmed that TTP-directed deadenylation principally relies on the CCR4–CAF1 complex. Indeed, recruitment of CAF1 seems to depend on the scaffolding protein NOT1, which interacts with the C-terminal domain of TTP, the presence of which is necessary for TTP-induced mRNA decay in HeLa cells [72]. Whether TTP directly interacts with these distinct proteins or is indirectly associated with them through a multi-protein complex remains to be determined. However, these three aforementioned studies all showed that recruitment of the CCR4–CAF1–NOT1 complex by TTP or its related proteins is mandatory for AMD and thereby highly contributes to this process [45, 46, 72]. Moreover, regulation of deadenylase recruitment has been shown to be dependent on the TTP phosphorylation status, reinforcing the notion that post-translational modifications of TTP family members are key events in the regulation of their activities [45, 46].

Deadenylation is a key step in AMD and it has been shown that poly(A) binding protein (PABP), which protects the mRNA poly(A) tail from degradation and is involved in mRNA circularization upon binding to eukaryotic translation initiation factor 4G (eIF4G), could indeed inhibit TTP-directed deadenylation [45, 73]. This TTP–PABP interaction has been further confirmed and characterized by yeast two-hybrid analyses and shown to be RNA-independent [74]. Therefore, it has been speculated that TTP binding to PABP can induce its displacement from the poly(A) tail of ARE-containing mRNAs and thereby promote 3′ to 5′degradation.

Once the poly(A) tail has been removed, further mRNA degradation can occur either in a 3′ to 5′ or in a 5′ to 3′ way. The exosome, a conserved protein complex with a 3′ to 5′ exonuclease activity is responsible for the 3′-end trimming of RNA and has been shown to be involved in AMD, among other functions [75, 76]. Exosome integrity is required for AMD and it has been suggested that components of the exosome can bind ARE-containing transcripts [76, 77]. Interestingly, association between the purified exosome complex and TTP has been detected and, in the same study, TTP appears to promote exosome-directed degradation of ARE-containing mRNAs [75]. Since then, interactions with several components of the exosome have been shown by co-immunoprecipitation. TTP has been reported to specifically recruit the functional exosome onto ARE-containing mRNAs and thereby to promote mRNA decay [78]. In addition to this 3′–5′ mRNA decay, ARE-containing transcripts are subjected to 5′–3′ degradation as well. TTP and TIS11b/BRF1 interact with decapping enzymes and the cytoplasmic 5′-3′exoribonuclease Xrn1, thereby promoting their recruitment onto the mRNA, mRNA decapping as well as 5′ mRNA degradation [71, 79]. It is difficult to determine the relative importance of 5′- versus 3′-AMD. Specific depletion of either the decapping enzyme Xrn1 or a component of the exosome complex inhibits AMD, suggesting the participation of both mechanisms in living cells [80].

Together, these studies support a model of mRNA stability regulation by TTP or its related proteins, where these zinc-finger proteins act as docking proteins for components of the mRNA decay machinery, as depicted in Fig. 3.

Fig. 3.

Involvement of TTP family proteins in the mRNA degradation pathway. Upon binding to ARE-containing transcripts, TTP proteins may interfere with mRNA circularization via its interaction with PABP. Subsequently, deadenylases are recruited onto the transcript either by direct interaction with TTP proteins (Crr4–Caf–Not complex) or indirectly (PARN). This step is modulated via TTP phosphorylation. Interactions of TTP and related proteins with decapping enzymes (Dcp1/2) may then promote decapping of the transcript, which can thereafter be trimmed by the 5′-exoribonuclease Xrn1. On the 3′-end, TTP proteins can also interact with the exosome complex which associates with the scavenger decapping enzyme (DcpS) to achieve 3′-decay of the transcript

Alternative functions of the TTP/TIS11 proteins throughout the life of an mRNA

In addition to their well-known effects on mRNA stability, emerging experimental evidence indicates that the TTP family of RNA binding proteins might participate in the regulation of almost all the key steps controlling mRNA from biogenesis to decay.

Functions of the TTP/TIS11 family members in mRNA transcription and 3′-end processing

At the onset of their discovery, because of the presence of a TZF domain in their sequence, it had been speculated that TTP and related proteins might be transcription factors. Two recent studies seem to eventually sustain this hypothesis. TIS11 possesses the potential to activate transcription when fused to the GAL4 DNA binding domain [81]. TIS11b/BRF1 was also shown to directly interact with the transcription factor HNF1α (hepatocyte nuclear factor 1α) as shown by pull-down assays, and this interaction seems to attenuate the HNF1α transcriptional activity [82].

The regulation of 3′-end mRNA processing and consecutive alternative polyadenylation is becoming an important challenge in RNA research as it appears that more than half of all human transcripts contain more than one poly(A) signal [83]. In yeast cells, Cth2, one of the homologues of human TTP and TIS11b, can interfere, beyond its activity in ARE-dependent degradation, with poly(A) site selection when the AREs are located in close proximity with the poly(A) signal [84]. Deletion of a specific domain of Cth2 allows the synthesis of a 3′-extended transcript, due to selection of a distal poly(A) signal in the target transcript.

Recent evidence has identified a similar mechanism in mammalian cells. In endothelial cells, TIS11b has been shown to modulate 3′-end processing of an ARE-containing mRNA (encoding Dll4) that contained TIS11b binding sites embedded in the actual poly(A) signal [27]. In these cells, specific inhibition of TIS11b, which appears to be localized in the nucleus, had no effect at all on mRNA stability, suggesting that the functions of TIS11b might highly depend on its nucleocytoplasmic localization. Since then, a high-throughput analysis has identified and characterized a direct interaction between TTP and poly(A)-binding protein nuclear 1 (PABPN1). This interaction induces the inhibition of the polyadenylation of an ARE-containing reporter transcript. Interestingly, this interaction seems to be dependent on the phosphorylation status of TTP with better PABPN1 binding for hypophosphorylated TTP [85].

Interference of TTP family members with mRNA cleavage and polyadenylation is a very new finding that deserves further characterization. It may be an important nuclear function for these nucleocytoplasmic shuttling proteins as it has been observed that regulation of 3′-UTR length (one of the main consequences of alternative polyadenylation) highly contributes to the control of gene expression [86].

Functions of the TTP/TIS11 family members in mRNA transport and/or localization

The subcellular compartment in which TTP or its related proteins are actually recruited onto ARE-containing mRNAs is not exactly known. At least in some circumstances, it has been suggested that this recruitment occurs in the nucleus. Indeed, the yeast TTP homolog Cth2 seems to be co-transcriptionally recruited onto nascent ARE-containing transcripts [27, 84, 87]. It has been shown that disruption of yeast Cth2 shuttling impaired AMD, suggesting a model whereby TTP and related proteins would be able to recruit mRNAs in the nucleus and deliver them to the cytoplasm for degradation [87]. These data suggested that co-transcriptional recruitment of these RBPs can occur and that yeast Cth2 might contribute to the nuclear export of mRNAs. Such a mechanism, however, remains to be validated in mammalian cells.

Following transcription, whether bound or unbound by TTP family members, mRNAs translocate to the cytoplasm where they are directed towards translation, storage, or decay. Processing bodies (PB) are RNA granules located in discrete cytoplasmic foci that contain decapping and 5′ to 3′ decay enzymes (Xrn1) and deadenylases among other proteins. These small structures constitute a specialized compartment for mRNA degradation given the promiscuity of multiple components of the decay machinery even though mRNA decay is not restricted to PBs [88]. As mentioned above, TTP can interact with decapping enzymes and exoribonucleases promoting transcript degradation and is found to be part of PBs [79, 89]. Actually, it has been shown that TTP and its related proteins can direct the localization of ARE-containing transcripts to PBs for decay and thereby can induce PB nucleation by aggregation of ribonucleoproteins [90].

The composition of PBs is highly dynamic, with transcripts and proteins able to move in and out of these structures. It has been shown that TTP and its related proteins can shuttle between PBs and stress granules (SG). SGs are aggregates of mRNAs stalled in the preinitation complex of ribosome assembly induced under stress conditions [89]. The presence of TTP and related proteins in SGs therefore suggests that they are involved in translation repression. Binding of TTP and its family members onto ARE-containing transcripts may allow escorting them to SGs or PBs for translation suppression or degradation, respectively, and may thereby contribute to mRNA sorting. TTP translocation between these RNA granules seems to rely on TTP interaction with transportin (TRN), a member of the importin-β family involved in nucleocytoplasmic transport of macromolecules [91]. TTP and its associated mRNAs interact with transportin, as it was shown that silencing of transportin sequesters TTP in PBs and therefore reduces their translocation to SGs. Moreover, the phosphorylation status of TTP modulates its binding to 14-3-3 protein and it was shown that formation of this molecular complex is able to exclude TTP and associated mRNAs from SGs [13]. These last two studies also showed that regulated trafficking of TTP between PBs and SGs induces an alteration of AMD, suggesting that TTP can select ARE-containing mRNAs stalled in translation in SGs to direct them to PBs for decay [13, 91]. These data argue in favor of a model suggesting that TTP and related proteins play a critical role in determining the fate of ARE transcripts depending on the environmental and stress conditions.

Functions of the TTP/TIS11 family members in mRNA translation

In addition to their role in mRNA decay, AREs have been known for a long time to be involved in the regulation of translation via the binding of specific AU-binding proteins such as TIA-1, TIAR-1, and HuR [92]. Furthermore, translational regulation of ARE-containing transcripts might be a means to control gene expression, in particular during serum starvation [93].

Indeed, an increasing number of studies suggest that TTP and its related proteins are involved in ARE-mediated regulation of translation under certain circumstances. First, following LPS stimulation, TTP can be found associated with polysomes in macrophages, suggesting a possible involvement in translation regulation [94, 95]. Second, a recent analysis showed that TTP specifically inhibits translation of an ARE-containing reporter transcript by excluding the targeted transcript from heavy polyribosomal fractions [96]. The same study further showed that TTP can associate directly with the helicase RCK/P54, which plays a major role in translation repression. Both this interaction and the helicase activity are necessary for ARE-mediated repression of translation. Interestingly, a previous study has shown a similar interaction in yeast between TTP and RCK/P54 homologues, supporting the idea that in yeast and mammals TTP-related proteins may have convergent functions even though translational inhibition has not been addressed in the latter report [97].

However, the involvement of TTP in ARE-dependent repression of translation is not clearly understood. A recent screening for TTP protein partners identified a specific interaction with Cullin 4B (Cul4B), which is a scaffolding component of a ubiquitin E3 ligase complex [98]. Depletion of Cul4B in macrophages decreased TNF-α mRNA stability and, in the meantime, inhibited its loading to polysomes upon LPS induction. The authors then suggested that recruitment of Cul4B to TTP promotes translation of the transcript, indicating that TTP function in translational regulation is more complex than anticipated and needs further characterization. Moreover, regulation of RNA translation is not a specific feature of TTP. Indeed, genetic disruption of TIS11b/BRF1 in mice revealed vascular defects and associated increased expression of VEGF, a potent angiogenic factor [16]. VEGF mRNA had been previously shown to be a target of both TIS11b and TTP, with a marked effect on mRNA stability observed in in vitro experiments [99–101]. However, very convincing data from the analysis of TIS11b^−/− fibroblasts showed that VEGF up-regulation in this in vivo situation was due to modification of mRNA translation efficiency and not related to changes in transcript stability [16]. Altogether, these data suggested that the physiological functions of TTP and related proteins might highly depend on the cell type and on the extracellular environment (hormones, metabolic nutrients, oxygenation, stress).

Functions of the TTP/TIS11 family members in microRNA-regulated pathways

mRNA 3′-UTRs are docking sites for both RNA-binding proteins and microRNAs (miRNAs), which both regulate stability and/or translation of the transcripts. Recent experimental data suggest numerous similarities and connections between the miRNA-regulated pathway and the ARE-mediated control of gene expression [102]. Concerning TTP and its related proteins, cooperation between TTP, miR-16, and AGO2, which are part of the RNA-induced silencing complex (RISC), has been described in ARE-mediated control of the TNF-α transcript [103]. This work identified a requirement for the miRNA pathway in TTP-mediated ARE-dependent decay. TTP cooperation with AGO2, without direct binding to miR-16, may assist the miRNA to specifically target the AU-rich element. This indicated that ARE-dependent decay may rely, at least partially, on the miRNA pathway. However, this interaction with miRNAs seems dispensable as depletion of components of the miRNA pathway does not prevent AMD in Drosophila and mouse cells [104]. Interestingly, the best evidence for a role played by TTP or related proteins in miRNA- or siRNA-regulated pathways came from an analysis in Drosophila cells, where only one TTP homologue is present [105]. A screening set up to identify factors involved in the RNAi pathway indicated that Drosophila TTP is required to achieve complete siRNA silencing, but the mechanism of this action remains to be characterized. Recently, a report showed that TTP could induce ARE-mediated decay of the Lin28 transcript, which is a negative modulator of the let7 microRNA, and thereby stimulate the biogenesis of the let7 microRNA in cancer cells [106].

Taken together, these studies suggest that the connection between ARE-dependent degradation and the miRNA pathway may be quite complex and needs further investigation.

RNA-binding independent functions of the TTP/TIS11 family members

In addition to the aforementioned functions of the TTP family members, recent data showed that, under some circumstances, their RNA-binding properties are not absolutely required for their activity. Two independent reports published back to back in 2009 identified TTP as a modulator of NF-κB signaling [107, 108]. Following NF-κB activation, TTP binding to the p65 subunit of this transcription factor seems able to impair either its nucleocytoplasmic shuttling or its acetylation and thereby to contribute to attenuation of NF-κB signaling.

Functions of TTP and related proteins in biological processes and diseases

Regulation of inflammation is one example of a biological process for which involvement of TTP and its related proteins is well described. As mentioned above, genetic inactivation of TTP in mice induces a complex inflammatory syndrome that is mainly due to TNF-α overproduction [66]. Following this study, granulocyte–macrophage colony-stimulating factor (GM-CSF) is also overexpressed in TTP^−/− macrophages, reinforcing the key player role of TTP in mediating the inflammatory response [109]. Moreover, a high proportion of ARE-containing RNAs are transcripts encoding proteins involved in immune functions and the list of mediators of inflammation experimentally validated as targets for ARE-mediated decay keeps growing (for review, see [48]). As a matter of fact, TTP regulates mRNA stability of both inflammatory and anti-inflammatory cytokines, attenuates NF-κB signaling, and thereby coordinately modulates inflammation.

Because of the vast diversity of their potential targets, it is not surprising to find alterations of TTP family member expression in cancer [110]. For instance, it has been shown that the level of TIS11b/BRF1 expression is associated with cisplatin sensitivity in head and neck squamous cell carcinoma and that specific silencing of TIS11b contributes to the development of cisplatin resistance [111]. In fact, converging data argue in favor of a tumor suppressor role for TTP and its related proteins. Indeed, overexpression of TTP in a v-H-ras-dependent mast cell tumor mouse model notably delayed tumor progression [112]. Recently, it was reported that TTP expression levels are inversely correlated with aggressiveness, metastatic potential, and resistance to antitumorigenic treatment in breast cancer [113]. Moreover, a direct role of ARE-mediated regulation in cancer progression has been firmly established by a study showing that inducible deletion of both TIS11b and TIS11d in thymus leads to almost 100 % of mice developing lymphoblastic leukemia [21]. This study is the first to show that malignant development could be a direct consequence of dysregulated AMD. Since then, loss of TTP and TIS11b expression has been shown to be a hallmark of Myc-induced malignancy in mice and it was shown that TTP overexpression could override this malignant state, clearly confirming its tumor suppressor function [114]. Very recent reviews analyze in depth the molecular mechanisms that link TTP and related proteins with cancer [48, 49, 115].

TIS11b/BRF1 genetic knock-out in mice induced numerous vascular defects, suggesting its direct involvement in angiogenesis [16]. The control of potent angiogenic factor expression by TTP proteins had been previously documented and the list of ARE-regulated transcripts related to angiogenesis regulation is constantly growing [99, 100, 116]. Beside its role in neovascularization, TIS11b is also involved in the maintenance of stem cell pluripotency. Indeed, in embryonic stem (ES) cells, TIS11b gene expression is under the control of the key transcriptions factors that are also known to control pluripotency and silencing of TIS11b expression, promoting cell differentiation into cardiomyocytes [117, 118].

Therapeutic potentials of the TTP/TIS11 proteins

As mentioned above, TTP and related proteins are involved in numerous biological processes during which their dysregulation might contribute to established pathologies. Due to the numerous ARE-containing mRNA targets involved in carcinogenesis, tumoral angiogenesis, and inflammation, enforced overexpression of TTP proteins in tumor cells has been tested as a novel anti-tumoral therapeutic approach and shown to efficiently slow down tumor growth in mouse tumorigenesis models [100, 101]. Similarly, in an experimental periodontitis mouse model, adenovirus-delivered TTP was reported to inhibit bone loss through simultaneous down-regulation of the expression of several inflammatory cytokines [119]. Altogether, these data provide a proof of concept that the mRNA-destabilizing properties of TTP family members can be used to design multi-target therapies of inflammatory diseases and cancer.

Concluding remarks: TTP proteins and AU-rich elements are working partners from the synthesis to the decay of an mRNA

Since the first description of the function of TTP in AMD, accumulating experimental data have sustained that this RNA-binding protein and its homologues are actually involved in many different biological processes. It has been suggested that TTP proteins belong to a family of negative feedback regulators that contribute to the attenuation of growth factor signaling and therefore contribute to the coordination of gene expression upon changes of the cellular environment [120]. Expression and regulation of TTP and related proteins are indeed highly dependent on the cell type considered and this might, at least in part, explain the major differences in the phenotypes of the different knock-out mice and the reason why, under some circumstances, the TTP family members can compensate for one another. As an example, some of the ARE-regulated transcripts which have been described as up-regulated in TTP^−/− fibroblasts are not modified in TIS11b/BRF1^−/− fibroblasts [121]. It is therefore essential to distinguish physiological targets for each TTP protein and to be aware that target specificity is dependent on the physiological context.

Adding to this complexity, it is now increasingly clear that the molecular function of this family of RNA-binding proteins is not restricted to AMD. Poly(A) site selection, translation regulation or mRNA transport are key steps in mRNA life, which are all regulated by TTP family members and will ultimately determine the fate of the transcript, as depicted in Fig. 4. AU-rich elements (or other yet-to-be-identified binding sites for the TTP proteins) will therefore mark transcripts for specific regulation. Depending on the local environment of this cis-regulating element, TTP proteins might trigger different mechanisms of regulation. Recruitment of TTP and related proteins within the poly(A) signal during transcription in the nucleus or in close proximity to an miRNA-binding site in the cytoplasm have different consequences in terms of transcript regulation.

Fig. 4.

Multiple functions of TTP proteins in mRNA life. TTP and TIS11b have been shown to modulate 3′-end mRNA maturation and poly(A) site selection in the cell nucleus. Recruitment of TTP proteins onto ARE-containing transcripts may occur under some circumstances in the nucleus and interactions with the nuclear pore complex may modulate mRNA delivery to the cytoplasm. After destabilization of the circularized mRNA, recruitment of deadenylases may direct AU-rich mRNAs to either 5′- or 3′-decay. TIS11 proteins are component of Processing Bodies (PB, in yellow) as well as Stress Granules (SG, in blue) induced in stress conditions. They are involved in PBs nucleation and promote 5′-mediated decay of target transcripts as well as their sorting and escorting between PBs and SG (interaction with transportin). TTP also regulates AU-rich mediated translation as it has been shown to exclude ARE-containing RNAs from heavy polysomes. Specific protein interactions with TTP seem to increase (Cul4B) or decrease (Rck/p54) polysome loading of ARE-containing transcripts. See text for detailed description of these and other functions of TTP/TIS11 proteins

The existence of such interconnected functions for a specific RNA-binding protein is now emerging as a general concept. The list of RNA-binding proteins with multiple functions keeps growing and recent high-throughput sequencing of binding sites for these proteins allowed identification and characterization of new functions. For example HuR, which was previously known as an mRNA stabilizing factor, has been reported to modulate mRNA splicing. Similarly, NOVA, previously described as a splicing factor, can modulate poly(A) site selection and alternative polyadenylation [122–124]. In light of these recent observations, one could predict that further analyses will define a broader range of regulatory functions for TTP proteins in the life of an mRNA than originally anticipated.